Gamma polyglutamated aminopterin and uses thereof

ABSTRACT

The disclosure relates generally to gamma polyglutamated aminopterin compositions, including delivery vehicles such as liposomes containing the gamma polyglutamated aminopterin, and methods of making and using the gamma polyglutamated aminopterin compositions to treat hyperproliferative disorders (e.g., cancer) and disorders of the immune system (e.g., inflammation and autoimmune diseases such as rheumatoid arthritis).

BACKGROUND

This disclosure generally relates to gamma polyglutamated aminopterincompositions, including delivery vehicles such as liposomes containingthe gamma polyglutamated aminopterin compositions, and methods of makingand using the compositions to treat diseases includinghyperproliferative diseases such as cancer, disorders of the immunesystem such as rheumatoid arthritis, and infectious diseases such as HIVand malaria. Aminopterin (AMT), orN-4-[[2,4-diamino-6-pteridinyl)-methyl]amino]benzoyl]-L-glutamic acid,is a potent antifolate that was one of the first antifolates developedand the first to demonstrate significant clinical efficacy. Aminopterinbears the closest structural similarity to folic acid, differing fromthe natural substrate by only two atoms. Aminopterin was used clinicallyas a single agent in the 1940s and 1950s for the treatment of acuteleukemia, psoriasis and arthritis in humans.

Based mainly on the results from animal studies and anecdotal humanexperience, its clinical use ceased in the mid-1950s when aminopterinwas determined to have inferior pharmacologic properties. Methotrexatewas perceived be less toxic and efficacious than aminopterin in treatingpsoriasis, however the doses compared were not equipotent to oneanother, with methotrexate being used in an amount 4-fold less thanwould be required to be equipotent with aminopterin (Rees and Bennett,Arch. Dermatol. 83:970-72, June 1961; and Strakosch, Dermatologica).Aminopterin was for the most part replaced by methotrexate around 1955,and methotrexate became a standard. (Sirotnal and Donsback, Cancer Res.32:2120, 1972; and Ratliff et al., J. Clin. Oneal. 16:1458, 1998). Inaddition to toxicity concerns, there were also there were also apparentchallenges with the manufacture of Aminopterin. In recent years howeverthere has been renewed interest in aminopterin because of the generalperception that it's utility in a variety of disease with unmet medicalneed has been under-explored. More recently the use of aminopterin inacute refractory leukemia has been revisited. (Cole P D, Drachtman etal. Cancer Chemother Pharmacol. 2008 June; 62(1):65-75). Aminopterin wascompared to methotrexate in patients with acute leukemia in patientswith leukemia. Treatment with aminopterin in leukemia when compared tomethotrexates lead to greater accumulation of the more activepolyglutamates derivatives of aminopterin than the polyglutamatederivatives of methotrexate. (Cole P D, Drachtman et al. Clin CancerRes. 2005 Nov. 15; 11(22):8089-96).

Potentially, many of the approved uses of methotrexate could be treatedusing aminopterin. The indications for methotrexate are summarized belowbecause these are potential conditions in which an improved formulationof aminopterin could play a role.

In the United States, Methotrexate is indicated in the treatment ofgestational choriocarcinoma, chorioadenoma destruens and hydatidiformmole. In acute lymphocytic leukemia, methotrexate is indicated in theprophylaxis of meningeal leukemia and is used in maintenance therapy incombination with other chemotherapeutic agents. Methotrexate is alsoindicated in the treatment of meningeal leukemia. Methotrexate is usedalone or in combination with other anticancer agents in the treatment ofbreast cancer, epidermoid cancers of the head and neck, advanced mycosisfungoides (cutaneous T cell lymphoma), and lung cancer, particularlysquamous cell and small cell types. Methotrexate is also used incombination with other chemotherapeutic agents in the treatment ofadvanced stage non-Hodgkin's lymphomas.

Methotrexate in high doses followed by leucovorin rescue in combinationwith other chemotherapeutic agents is effective in prolongingrelapse-free survival in patients with nonmetastatic osteosarcoma whohave undergone surgical resection or amputation for the primary tumor.

Methotrexate is approved for the symptomatic control of severe,recalcitrant, disabling psoriasis that is not adequately responsive toother forms of therapy, but only when the diagnosis has beenestablished, as by biopsy and/or after dermatologic consultation. It isimportant to ensure that a psoriasis “flare” is not due to anundiagnosed concomitant disease affecting immune responses. Aminopterinin previous studies showed very promising activities against psoriasis(Gubner, Arch. Derm. 64:688, 1951; Rees et al., Arch. Derm., 90:544,1964; and Gubner et al., Am. J. Med. Sci. 22:176, 1951).

Methotrexate is approved in the management of selected adults withsevere, active rheumatoid arthritis (ACR criteria), or children withactive polyarticular-course juvenile rheumatoid arthritis, who have hadan insufficient therapeutic response to, or are intolerant of, anadequate trial of first-line therapy including full dose non-steroidalanti-inflammatory agents (NSAIDs).

Over time, methotrexate has achieved widespread clinical use as anessential component of multidrug regimens for treating acutelymphoblastic leukemia (ALL), lymphomas, and solid tumors worldwide.Methotrexate (MTX) is also the anchor-drug most widely applied diseasemodifying antirheumatic drug (DMARD) in the treatment of patients withrheumatoid arthritis (RA). It is often used either as single agent or incombination with other DMARDs (e.g., sulfasalazine andhydroxychloroquine) and MTX use is obligate in most treatment strategiesinvolving biological agents (e.g., anti-TNFα and anti CD20 monoclonalantibodies. Used in the treatment of breast, advanced head and neck,lung, and stomach cancers, osteosarcoma, Non-Hodgkin's lymphoma (NHL),acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-celllymphoma) choriocarcinoma, and chorioadenoma. Off-label cancer uses formethotrexate also include nonleukemic meningeal cancer, soft tissuesarcoma (desmoid tumors, aggressive fibromatosis), bladder cancer,Central Nervous System (CNS) lymphoma, and prevention ofgraft-versus-host disease.

MTX is also used in non-cancerous conditions such as psoriasis andrheumatoid arthritis, inflammatory bowel disease (IBD), systemicinflammation, atherosclerosis, cardiovascular disease (CVD), coronaryartery disease, and gestational trophoblastic diseases. Some off-labelnon-cancer uses include Crohn disease, dermatomyositis/polymyositis,ectopic pregnancy, systemic lupus erythematosus, and Takayasu arteritis.

Methotrexate is a folate analog that differs from the folate by thesubstitution of an amino group for a hydroxyl at the 4-position of thepteridine ring. This minor structural alteration results in the abilityof MTX to inhibit the active catalytic site of dihydrofolate reductase(DHFR) which catalyzes the production of tetrahydrofolate (THF) fromdihydrofolate (DHF). Consequently, methotrexate interferes with thesynthesis of tetrahydrofolate (THF), which serves as the primaryone-carbon carrier for enzymatic processes involved in de novo synthesisof thymidylate, purine nucleotides, and the amino acids serine andmethionine. The inhibition of these metabolic processes disrupt theformation of DNA, RNA, and key cellular proteins.

Folate is an essential cofactor that mediates the transfer of one-carbonunits involved in nucleotide biosynthesis and DNA repair, theremethylation of homocysteine (Hcy), and the methylation of DNA,proteins, and lipids. The only circulating forms of folates in the bloodare monoglutamates and folate monoglutamates are the only form of folatethat is transported across the cell membrane—likewise, the monoglutamateform of polyglutamatable antifolates such as aminopterin, aretransported across the cell membrane. Once taken up into cells,intracellular folate is converted to polyglutamates by the enzymefolylpoly-gamma-glutamate synthetase (FPGS).

Like other antifolates, aminopterin is transported into cells by thereduced folate carrier (RFC) system and folate receptors (FRs) α and βand by Proton Coupled Folate Transporter (PCFT) that is generally mostactive in a lower pH environment. RFC is the main transporter ofaminopterin at physiologic pH and is ubiquitously expressed in bothnormal and diseased cells. Consequently, aminopterin treatment oftensuffers from the dose-limiting toxicity that is a major obstacle incancer chemotherapy. Once inside the cell, aminopterin is polyglutamatedby FPGS, which may add up to 6 L-glutamyl groups in a L-gamma carboxylgroup linkage to the aminopterin. The L-gamma polyglutamation ofaminopterin by FPGS serves at least 2 main therapeutic purposes: (1) itgreatly enhances aminopterin affinity and inhibitory activity for DHFR;and (2) it facilitates the accumulation of polyglutamated aminopterin,which unlike aminopterin (monoglutamate), is not easily transported outof cells by cell efflux pumps.

While targeting folate metabolism and nucleotide biosynthesis is a wellestablished therapeutic strategy for cancer, for antifolates such asaminopterin, clinical efficacy is limited by a lack of tumor selectivityand the presence of de novo and acquired drug resistance. Like otherantifolates, aminopterin acts during DNA and RNA synthesis, andconsequently has a greater toxic effect on rapidly dividing cells suchas malignant and myeloid cells. Myelosuppression is typically thedose-limiting toxicity of aminopterin therapy and has limited theclinical applications of aminopterin.

Resistance to antifolates like aminopterin therapy is typicallyassociated with one or more of, (a) increased cell efflux pump activity,(b) decreased transport of AMT into cells (c) increased DHFR activity,(d) decreased folylpolyl-gamma-glutamate synthetase (FPGS) activity, and(e) increased gamma-glutamyl hydrolase (GGH) activity, which cleavesgamma polyglutamate chains attached to folates and antifolates.

The challenge to the longstanding (>30 years) observation thathigher-level polyglutamates of various antifolates have much greaterpotency compared to lower-level glutamates, has been that the scientificcommunity has relied on the intracellular FPGS mediated mechanisms toconvert the lower-level glutamates to their higher-level forms. Thepresent inventions provide the means to deliver higher-levelpolyglutamate forms of antifolates including aminopterin directly intothe cell, without having to rely on the cells machinery to achieve thisgoal.

The provided gamma polyglutamated aminopterin compositions deliver astrategy for overcoming the pharmacological challenges associated withthe dose limiting toxicities and with treatment resistance associatedwith aminopterin therapy. The provided methods deliver to cancer cells anovel gamma polyglutamated form of aminopterin while (1)minimizing/reducing exposure to normal tissue cells, (2)optimizing/improving the cytotoxic effect of aminopterin-based agents oncancer cells and (3) minimizing/reducing the impact of the efflux pumps,and other resistance mechanisms that limit the therapeutic efficacy ofaminopterin.

BRIEF SUMMARY

This disclosure generally relates gamma polyglutamated aminopterin (AMN)compositions and methods of making and using the compositions to treatdiseases including hyperproliferative diseases such as cancer, disordersof the immune system such as inflammation and rheumatoid arthritis, andinfectious disease such as HIV and malaria.

In some embodiments, the disclosure provides:

-   [1] A composition comprising a gamma polyglutamated aminopterin;-   [2] the composition of [1], wherein the gamma polyglutamated    aminopterin comprises 1-10 glutamyl groups having gamma carboxyl    group linkages;-   [3] the composition of [1] or [2], wherein the gamma polyglutamated    aminopterin contains 4, 5, 2-10, 4-6, or more than 5, glutamyl    groups having gamma carboxyl group linkages;-   [4] the composition according to any of [1]-[3], wherein the gamma    polyglutamated aminopterin is gamma tetraglutamated aminopterin;-   [5] the composition according to any of [1]-[3], wherein the gamma    polyglutamated aminopterin is gamma pentaglutamated aminopterin;-   [6] the composition according to any of [1]-[3], wherein the gamma    polyglutamated aminopterin is gamma hexaglutamated aminopterin;-   [7] the composition according to any of [1]-[6], wherein    -   (a) the gamma polyglutamated aminopterin comprises two or more        glutamyl groups in the L-form having gamma carboxyl group        linkages,    -   (b) each of the glutamyl groups of the gamma polyglutamated        aminopterin is in the L-form and has a gamma carboxyl group        linkage;    -   (c) at least one of the glutamyl groups of the gamma        polyglutamated aminopterin is in the D-form and has a gamma        carboxyl group linkage,    -   (d) each of the glutamyl groups of the gamma polyglutamated        aminopterin other than the glutamyl group of aminopterin is in        the D-form and has a gamma carboxyl group linkage, or    -   (e) the gamma polyglutamated aminopterin comprises two or more        glutamyl groups in the L-form and at least one glutamyl group in        the D-form having gamma carboxyl group linkages;-   [8] the composition according to [4], wherein (a) each of the    glutamyl groups is in the L-form and has a gamma carboxyl group    linkage or (b) each of the glutamyl groups other than the glutamyl    group of aminopterin is in the D-form and each of the glutamyl    groups has a gamma carboxyl group linkage;-   [9] the composition of [5], wherein (a) each of the glutamyl groups    is in the L-form and has a gamma carboxyl group linkage or (b) each    of the glutamyl groups other than the glutamyl group of aminopterin    is in the D-form and each of the glutamyl groups has a gamma    carboxyl group linkage;-   [10] the composition of [6], wherein (a) each of the glutamyl groups    is in the L-form and has a gamma carboxyl group linkage or (b) each    of the glutamyl groups other than the glutamyl group of aminopterin    is in the D-form and each of the glutamyl groups has a gamma    carboxyl group linkage;-   [11] the composition according to any of [1]-[10], wherein the gamma    polyglutamated aminopterin is polyglutamable by FGPS under    physiological conditions and/or wherein the polyglutamated AMN has a    lower uptake rate (<30%) by hepatic cells than AMN;-   [12] a liposomal composition comprising the gamma polyglutamated    aminopterin according to any of [1]-[11] (Lp-γPAMN);-   [13] the Lp-γPAMN composition according to [12], wherein the gamma    polyglutamated aminopterin comprises two or more glutamyl groups in    the L-form;-   [14] the Lp-γPAMN composition according to [12] or [13], wherein    each of the glutamyl groups of the gamma polyglutamated aminopterin    is in the L-form;-   [15] the Lp-γPAMN composition of [12] or [13], wherein at least one    of the glutamyl groups of the gamma polyglutamated aminopterin is in    the D-form;-   [16] the Lp-γPAMN composition according to any of [12]-[15], wherein    the liposome comprises a gamma polyglutamated aminopterin comprising    1-10 glutamyl groups having gamma carboxyl group linkages;-   [17] the Lp-γPAMN composition according to any of [12]-[16], wherein    the liposome comprises a gamma polyglutamated aminopterin containing    4, 5, 2-10, 4-6, or more than 5, glutamyl groups;-   [18] the Lp-γPAMN composition according to any of [12]-[17], wherein    the liposome comprises gamma tetraglutamated aminopterin;-   [19] the Lp-γPAMN composition according to any of [12]-[17], wherein    the liposome comprises gamma pentaglutamated aminopterin;-   [20] The Lp-γPAMN composition according to any of [12]-[17], wherein    the liposome comprises gamma hexaglutamated aminopterin;-   [21] the Lp-γPAMN composition according to any of [12]-[20], wherein    the liposome is not pegylated (PγLp-γPAMN);-   [22] the Lp-γPAMN composition according to any of [12]-[20], wherein    the liposome is pegylated (PγLp-γPAMN);-   [23] the Lp-γPAMN composition according to any of [12]-[22], wherein    the liposomes comprise at least 1% weight by weight (w/w) of the    gamma polyglutamated aminopterin or wherein during the process of    preparing the Lp-γPAMN, at least 1% of the starting material of    gamma polyglutamated AMN is encapsulated (entrapped) in the    Lp-γPAMN;-   [24] the Lp-γPAMN composition according to any of [12]-[23], wherein    the liposome has a diameter in the range of 20 nm to 500 nm;-   [25] the Lp-γPAMN composition according to any of [12]-[24], wherein    the liposome has a diameter in the range of 20 nm to 200 nm;-   [26] the Lp-γPAMN composition according to any of [12]-[25], wherein    the liposome has a diameter in the range of 80 nm to 120 nm;-   [27] the Lp-γPAMN composition according to any of [12]-[26], wherein    the liposome is formed from liposomal components;-   [28] the Lp-γPAMN composition according to [27], wherein the    liposomal components comprise at least one of an anionic lipid and a    neutral lipid;-   [29] the Lp-γPAMN composition according to [27] or [28], wherein the    liposomal components comprise at least one selected from the group    consisting of: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG;    cholesterol; cholesterol-PEG; and cholesterol-maleimide;-   [30] the Lp-γPAMN composition according to any of [27]-[29], wherein    the liposomal components comprise at least one selected from the    group consisting of: DSPE; DSPE-PEG; DSPE-PEG-FITC;    DSPE-PEG-maleimide; cholesterol; and HSPC;-   [31] the Lp-γPAMN composition according to any of [27]-[30], wherein    one or more liposomal components further comprises a steric    stabilizer;-   [32] the Lp-γPAMN composition according to [31], wherein the steric    stabilizer is at least one selected from the group consisting of    polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside    (GM1); poly(vinyl pyrrolidone) (PVP); poly(acrylamide) (PAA);    poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl    polyglycerol; poly[N-(2-hydroxypropyl) methacrylamide]; amphiphilic    poly-N-vinylpyrrolidones; L-amino-acid-based polymer; oligoglycerol,    copolymer containing polyethylene glycol and polypropylene oxide,    Poloxamer 188, and polyvinyl alcohol;-   [33] the Lp-γPAMN composition according to [32], wherein the steric    stabilizer is PEG and the PEG has a number average molecular weight    (Mn) of 200 to 5000 daltons;-   [34] the Lp-γPAMN composition according to any of [12]-[33], wherein    the liposome is anionic or neutral;-   [35] the Lp-γPAMN composition according to any of [12]-[33], wherein    the liposome has a zeta potential that is less than or equal to    zero;-   [36] the Lp-γPAMN composition according to any of [12]-[33], wherein    the liposome has a zeta potential that is between 0 to −150 mV;-   [37] the Lp-γPAMN composition according to any of [12]-[33], wherein    the liposome has a zeta potential that is between −30 to −50 mV;-   [38] the Lp-γPAMN composition according to any of [12]-[33], wherein    the liposome is cationic;-   [39] the Lp-γPAMN composition according to any of [12]-[38], wherein    the liposome has an interior space comprising the gamma    polyglutamated aminopterin and an aqueous pharmaceutically    acceptable carrier;-   [40] the Lp-γPAMN composition of [39], wherein the pharmaceutically    acceptable carrier comprises a tonicity agent such as dextrose,    mannitol, glycerine, potassium chloride, sodium chloride, at a    concentration of greater than 1%;-   [41] the Lp-γPAMN composition of [39], wherein the aqueous    pharmaceutically acceptable carrier is trehalose;-   [42] the Lp-γPAMN composition of [41], wherein the pharmaceutically    acceptable carrier comprises 1% to 50% trehalose;-   [43] the Lp-γPAMN composition according to any of [39]-[42], wherein    the pharmaceutically acceptable carrier comprises 1% to 50% dextrose    solution;-   [44] the Lp-γPAMN composition according to any of [39]-[43], wherein    the interior space of the liposome comprises 5% dextrose suspended    in an HEPES buffered solution;-   [45] the Lp-γPAMN composition according to any of [39]-[44], wherein    the pharmaceutically acceptable carrier comprises a buffer such as    HEPES Buffered Saline (HBS) or similar, at a concentration of    between 1 to 200 mM and a pH of between 2 to 8;-   [46] the Lp-γPAMN composition according to any of [39]-[45], wherein    the pharmaceutically acceptable carrier comprises a total    concentration of sodium acetate and calcium acetate of between 50 mM    to 500 mM;-   [47] the Lp-γPAMN composition according to any of [12]-[46], wherein    the interior space of the liposome has a pH of 5-8 or a pH of 6-7,    or any range therein between;-   [48] the Lp-γPAMN composition according to any of [12]-[47], wherein    the liposome comprises less than 500,000 or less than 200,000    molecules of the gamma polyglutamated aminopterin;-   [49] the Lp-γPAMN composition according to any of [12]-[48], wherein    the liposome comprises between 10 to 100,000 molecules of the gamma    polyglutamated aminopterin, or any range therein between;-   [50] the Lp-γPAMN composition according to any of [12]-[49], which    further comprises a targeting moiety and wherein the targeting    moiety has a specific affinity for a surface antigen on a target    cell of interest;-   [51] the Lp-γPAMN composition according to [50], wherein the    targeting moiety is attached to one or both of a PEG and the    exterior of the liposome, optionally wherein targeting moiety is    attached to one or both of the PEG and the exterior of the liposome    by a covalent bond;-   [52] the Lp-γPAMN composition of [50] or [51], wherein the targeting    moiety is a polypeptide;-   [53] the Lp-γPAMN composition according to any of [50]-[52], wherein    the targeting moiety is an antibody or an antigen binding fragment    of an antibody;-   [54] the Lp-γPAMN composition according to any of [50]-[53], wherein    the targeting moiety binds the surface antigen with an equilibrium    dissociation constant (Kd) in a range of 0;5×10⁻¹⁰ to 10×10⁻⁶ as    determined using BIACORE analysis;-   [55] the Lp-γPAMN composition according to any of [50]-[54], wherein    the targeting moiety specifically binds one or more folate receptors    selected from the group consisting of: folate receptor alpha (FR-α),    folate receptor beta (FR-β), and folate receptor delta (FR-δ);-   [56] the Lp-γPAMN composition according to any of [50]-[55], wherein    the targeting moiety comprises one or more selected from the group    consisting of: an antibody, a humanized antibody, an antigen binding    fragment of an antibody, a single chain antibody, a single-domain    antibody, a bi-specific antibody, a synthetic antibody, a pegylated    antibody, and a multimeric antibody;-   [57] the Lp-γPAMN composition according to any of [50]-[56], wherein    each pegylated liposome comprises from 1 to 1000 or 30-200 targeting    moieties;-   [58] the Lp-γPAMN composition according to any of [39]-[57], further    comprising one or more of an immunostimulatory agent, a detectable    marker and a maleimide, wherein the immunostimulatory agent, the    detectable marker or the maleimide is attached to said PEG or the    exterior of the liposome;-   [59] the Lp-γPAMN composition of [58], wherein the immunostimulating    agent is at least one selected from the group consisting of: a    protein immunostimulating agent; a nucleic acid immunostimulating    agent; a chemical immunostimulating agent; a hapten; and an    adjuvant;-   [60] the Lp-γPAMN composition of [58] or [59], wherein the    immunostimulating agent is at least one selected from the group    consisting of: a fluorescein; a fluorescein isothiocyanate (FITC); a    DNP; a beta glucan; a beta-1,3-glucan; a beta-1,6-glucan; a resolvin    (e.g., a Resolvin D such as D_(n-6DPA) or D_(n-3DPA), a Resolvin E,    or a T series resolvin); and a Toll-like receptor (TLR) modulating    agent such as, an oxidized low-density lipoprotein (e.g., OXPAC,    PGPC), and an eritoran lipid (e.g., E5564);-   [61] the Lp-γPAMN composition according to any of [58]-[60], wherein    the immunostimulatory agent and the detectable marker is the same;-   [62] the Lp-γPAMN composition according to any of [58]-[61], further    comprising a hapten;-   [63] the Lp-γPAMN composition of [62], wherein the hapten comprises    one or more of fluorescein or Beta 1, 6-glucan;-   [64] the Lp-γPAMN composition according to any of [12]-[63], which    further comprises at least one cryoprotectant selected from the    group consisting of mannitol; trehalose; sorbitol; and sucrose;-   [65] a targeted composition comprising the composition according to    any of [1]-[64];-   [66] a non-targeted composition comprising the composition according    to any of [1]-[49];-   [67] the Lp-γPAMN composition according to any of [12]-[66], which    further comprises carboplatin and/or pembroluzumab;-   [68] A pharmaceutical composition comprising the liposomal gamma    polyglutamated aminopterin composition according to any of    [12]-[67];-   [69] a pharmaceutical composition comprising gamma polyglutamated    aminopterin composition according to any of [1]-[7];-   [70] the composition of any of [1]-[69], for use in the treatment of    disease;-   [71] Use of the composition of any of [1]-[70], in the manufacture    of a medicament for the treatment of disease;-   [72] a method for treating or preventing disease in a subject    needing such treatment or prevention, the method comprising    administering the composition of any of [1]-[70] to the subject;-   [73] a method for treating or preventing disease in a subject    needing such treatment or prevention, the method comprising    administering the liposomal gamma polyglutamated aminopterin    composition of any of [12]-[69] to the subject;-   [74] a method of killing a hyperproliferative cell that comprises    contacting a hyperproliferative cell with the composition of any of    [1]-[69];-   [75] a method of killing a hyperproliferative cell that comprises    contacting a hyperproliferative cell with the liposomal gamma    polyglutamated aminopterin composition of any of [12]-[69];-   [76] the method of [74] or [75], wherein the hyperproliferative cell    is a cancer cell, a mammalian cell, and/or a human cell;-   [77] a method for treating cancer that comprises administering an    effective amount of the composition of any of [1]-[69] to a subject    having or at risk of having cancer;-   [78] a method for treating cancer that comprises administering an    effective amount of the liposomal gamma polyglutamated aminopterin    composition of any of [12]-[68] to a subject having or at risk of    having cancer;-   [79] the method of [77] or [78], wherein the cancer is selected from    the group consisting of: a non-hematologic malignancy including such    as for example, lung cancer, pancreatic cancer, breast cancer,    ovarian cancer, prostate cancer, head and neck cancer, gastric    cancer, gastrointestinal cancer, colorectal cancer, esophageal    cancer, cervical cancer, liver cancer, kidney cancer, biliary duct    cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,    osteosarcoma), brain cancer, central nervous system cancer, and    melanoma; and a hematologic malignancy such as for example, a    leukemia, a lymphoma and other B cell malignancies, myeloma and    other plasma cell dyscrasias;-   [80] the method of [77] or [78], wherein the cancer is a member    selected from the group consisting of: lung cancer, breast cancer,    colon cancer, pancreatic cancer, gastric cancer, bladder cancer,    head and neck cancer, ovarian cancer, and cervical cancer;-   [81] the method of [77] or [78], wherein the cancer is a member    selected from the group consisting of: colorectal cancer, lung    cancer, breast cancer, head and neck cancer, and pancreatic cancer;-   [82] the method of [77] or [78], wherein the cancer is selected from    the group consisting of: colorectal cancer, breast cancer, ovarian    cancer, lung cancer, head and neck cancer, pancreatic cancer,    gastric cancer, and mesothelioma;-   [83] a method for treating cancer that comprises administering an    effective amount of the Lp-γPAMN composition of any of [50]-[66] to    a subject having or at risk of having a cancer cell that expresses    on its surface a folate receptor bound by the targeting moiety;-   [84] a maintenance therapy for subjects that are undergoing or have    undergone cancer therapy that comprise administering an effective    amount of the composition of any of [1]-[69] to a subject that is    undergoing or has undergone cancer therapy;-   [85] a maintenance therapy for subjects that are undergoing or have    undergone cancer therapy that comprise administering an effective    amount of the liposomal gamma polyglutamated aminopterin composition    of any of [12]-[69] to a subject that is undergoing or has undergone    cancer therapy;-   [86] a method for treating a disorder of the immune system that    comprises administering an effective amount of the composition of    any of [1]-[69] to a subject having or at risk of having a disorder    of the immune system, optionally wherein the disorder of the immune    system is selected from: inflammation (e.g., acute and chronic),    systemic inflammation, rheumatoid arthritis, inflammatory bowel    disease (IBD), Crohn disease, dermatomyositis/polymyositis, systemic    lupus erythematosus, and Takayasu, and psoriasis;-   [87] a method for treating a disorder of the immune system that    comprises administering an effective amount of the liposomal gamma    polyglutamated aminopterin composition of any of [8]-[69] to a    subject having or at risk of having a disorder of the immune system,    optionally wherein the disorder of the immune system is selected    from: inflammation (e.g., acute and chronic), systemic inflammation,    rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn    disease, dermatomyositis/polymyositis, systemic lupus erythematosus,    and Takayasu, and psoriasis;-   [88] a method for treating:    -   (a) an infectious disease that comprises administering an        effective amount of the composition according to any of [1]-[69]        to a subject having or at risk of having an infectious disease;    -   (b) an infectious disease, cardiovascular disease, metabolic        disease, or another disease, that comprises administering an        effective amount of the composition according to of any of any        of [1]-[69] to a subject having or at risk of having an        infectious disease, cardiovascular disease, or another disease,        wherein the disease is a member selected from: atherosclerosis,        cardiovascular disease (CVD), coronary artery disease,        myocardial infarction, stroke, metabolic syndrome, a gestational        trophoblastic disease, and ectopic pregnancy;    -   (c) an autoimmune disease, that comprises administering an        effective amount of the composition according to of any of any        of [1]-[69] to a subject having or at risk of having an        autoimmune disease;    -   (d) rheumatoid arthritis, that comprises administering an        effective amount of the composition according to of any of any        of [1]-[69] to a subject having or at risk of having rheumatoid        arthritis;    -   (e) an inflammatory condition that comprises administering an        effective amount of the composition according to of any of any        of [1]-[69] to a subject having or at risk of having        inflammation, optionally wherein the inflammation is acute,        chronic, and/or systemic inflammation; or-   (f) a skin condition that comprises administering an effective    amount of the composition according to of any of claims any of    [1]-[69] to a subject having or at risk of having a skin condition,    optionally wherein the skin condition is psoriasis;-   [89] a method for treating an infectious disease that comprises    administering an effective amount of the liposomal gamma    polyglutamated aminopterin composition of any of [12]-[69] to a    subject having or at risk of having an infectious disease;-   [90] a method of delivering gamma polyglutamated aminopterin to a    tumor expressing a folate receptor on its surface, the method    comprising: administering the Lp-γPAMN composition of any of    [1]-[69] to a subject having the tumor in an amount to deliver a    therapeutically effective dose of the gamma polyglutamated    aminopterin to the tumor;-   [91] a method of preparing a gamma polyglutamated aminopterin    composition comprising the liposomal gamma polyglutamated    aminopterin composition of any of [12]-[69], the method comprising:    forming a mixture comprising: liposomal components and gamma    polyglutamated antifolate in solution; homogenizing the mixture to    form liposomes in the solution; and processing the mixture to form    liposomes containing gamma polyglutamated aminopterin;-   [92] a method of preparing the composition of any of [12]-[69]    comprising the steps of: forming a mixture comprising: liposomal    components and gamma polyglutamated aminopterin in a solution;    homogenizing the mixture to form liposomes in the solution;    processing the mixture to form liposomes entrapping and/or    encapsulating gamma polyglutamated aminopterin; and providing the    targeting moiety on a surface of the liposomes, the targeting moiety    having specific affinity for at least one of folate receptor alpha    (FR-α), folate receptor beta (FR-β) and folate receptor delta    (FR-δ);-   [93] the method according to [92], wherein the processing step    includes one or more steps of: thin film hydration, extrusion,    in-line mixing, ethanol injection technique, freezing-and-thawing    technique, reverse-phase evaporation, dynamic high pressure    microfluidization, microfluidic mixing, double emulsion,    freeze-dried double emulsion, 3D printing, membrane contactor    method, and stirring; and/or-   [94] the method according to [92], wherein said processing step    includes one or more steps of modifying the size of the liposomes by    one or more of steps of extrusion, high-pressure microfluidization,    and/or sonication;

In some embodiments, the disclosure provides a gamma polyglutamatedaminopterin (γPAMN) composition wherein at least 2 of the glutamylresidues of the gamma polyglutamated aminopterin have a gamma carboxylgroup linkage. In some embodiments, the γPAMN contains 2-20, 2-15, 2-10,2-5, or more than 5, glutamyl groups (including the glutamyl group inaminopterin). In some embodiments, the γPAMN comprises two or moreglutamyl groups in the L-form. In other embodiments, the γPAMN comprisesa glutamyl group in the D-form. In further embodiments, the γPAMNcomprises a glutamyl group in the D-form and two or more glutamyl groupsin the L-form.

In one embodiment, the γPAMN composition contains a chain of 3 glutamylgroups attached to the glutamyl group of aminopterin (i.e., atetraglutamated aminopterin). In some embodiments, the tetraglutamatedAMN comprises two or more glutamyl groups in the L-form. In otherembodiments, the tetraglutamated AMN comprises a glutamyl group in theD-form. In further embodiments, the tetraglutamated AMN comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form.

In one embodiment, the γPAMN composition contains a chain of 4γ-glutamyl groups attached to the glutamyl group of aminopterin (e.g.,γ-pentaglutamated aminopterin). In some embodiments, the gammapentaglutamated AMN comprises two or more glutamyl groups in the L-form.In other embodiments, the gamma pentaglutamated AMN comprises a glutamylgroup in the D-form. In further embodiments, the gamma pentaglutamatedAMN comprises a glutamyl group in the D-form and two or more glutamylgroups in the L-form.

In one embodiment, the γPAMN composition contains a chain of 5γ-glutamyl groups attached to the glutamyl group of aminopterin (e.g.,γ-hexaglutamated aminopterin). In some embodiments, the gammahexaglutamated AMN comprises two or more glutamyl groups in the L-form.In other embodiments, the gamma hexaglutamated AMN comprises a glutamylgroup in the D-form. In further embodiments, the gamma hexaglutamatedAMN comprises a glutamyl group in the D-form and two or more glutamylgroups in the L-form.

In additional embodiments, the disclosure provides compositionscontaining delivery vehicles such as liposomes filled with (e.g.,encapsulating) and/or otherwise associated with gamma polyglutamatedaminopterin, and methods of making and using the γPAMN filled/associateddelivery vehicle compositions (DV-γPAMN) to deliver gamma polyglutamatedaminopterin to diseased (e.g., cancerous) and/or targeted cells. Thesecompositions have uses that include but are not limited to treatingdiseases that include for example, hyperproliferative diseases such ascancer, disorders of the immune system such as inflammation andrheumatoid arthritis, and infectious disease such as HIV and malaria. Insome embodiments, gamma polyglutamated aminopterin in the DV-γPAMNcontains 2-20, 2-15, 2-10, 2-5, more than 5, or more than 20, glutamylgroups (including the glutamyl group in aminopterin). The DV-γPAMNfilled/associated delivery vehicle compositions provide improvements tothe efficacy and safety of delivering aminopterin to cancer cells byproviding the preferential delivery of a more cytotoxic payload (e.g.,polyglutamated aminopterin) compared to the cytotoxicity of aminopterinadministered in its monoglutamate state (AMN).

In additional embodiments, the disclosure provides a compositioncomprising a liposome encapsulating (filled with) gamma polyglutamatedaminopterin (Lp-γPAMN). In some embodiments, the gamma polyglutamatedaminopterin in the Lp-γPAMN contains 2-20, 2-15, 2-10, 2-5, or more than20, glutamyl groups (including the glutamyl group in aminopterin). Insome embodiments, the gamma polyglutamated aminopterin in the Lp-γPAMNcomprises two or more glutamyl groups in the L-form. In otherembodiments, the gamma polyglutamated aminopterin in the Lp-γPAMNcomprises a glutamyl group in the D-form. In further embodiments, thegamma polyglutamated aminopterin in the Lp-γPAMN comprises a glutamylgroup in the D-form and two or more glutamyl groups in the L-form.

In one embodiment, the Lp-γPAMN composition comprises a gammapolyglutamated AMN that contains a chain of 3 glutamyl groups attachedto the glutamyl group of aminopterin (i.e., tetraglutamatedaminopterin). In some embodiments, the tetraglutamated AMN comprises twoor more glutamyl groups in the L-form. In other embodiments, thetetraglutamated AMN comprises a glutamyl group in the D-form. In furtherembodiments, the tetraglutamated AMN comprises a glutamyl group in theD-form and two or more glutamyl groups in the L-form.

In one embodiment, the Lp-γPAMN composition comprises a gammapolyglutamated AMN that contains a chain of 4 γ-glutamyl groups attachedto the glutamyl group of aminopterin (e.g., γ-pentaglutamatedaminopterin). In some embodiments, the gamma pentaglutamated AMNcomprises two or more glutamyl groups in the L-form. In otherembodiments, the gamma pentaglutamated AMN comprises a glutamyl group inthe D-form. In further embodiments, the gamma pentaglutamated AMNcomprises a glutamyl group in the D-form and two or more glutamyl groupsin the L-form.

In one embodiment, the Lp-γPAMN composition comprises a gammapolyglutamated AMN that contains a chain of 5 γ-glutamyl groups attachedto the glutamyl group of aminopterin (e.g., γ-hexaglutamatedaminopterin). In some embodiments, the gamma hexaglutamated AMNcomprises two or more glutamyl groups in the L-form. In otherembodiments, the gamma hexaglutamated AMN comprises a glutamyl group inthe D-form. In further embodiments, the gamma hexaglutamated AMNcomprises a glutamyl group in the D-form and two or more glutamyl groupsin the L-form.

In some embodiments, the Lp-γPAMN composition is cationic. In someembodiments, the Lp-γPAMN liposome is cationic and has a diameter in therange of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to150 nm, or any range therein between. In further embodiments, theLp-γPAMN liposome is cationic and the composition has a diameter in therange of 80 nm to 120 nm, or any range therein between. In someembodiments, the cationic Lp-γPAMN composition comprises at least 1%,5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,or more than 75%, w/w of the gamma polyglutamated AMN. In someembodiments, during the process of preparing the Lp-γPAMN, at least 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, or more than 75%, of the starting material of gamma polyglutamatedAMN is encapsulated (entrapped) in the cationic Lp-γPAMN. In additionalembodiments, the gamma polyglutamated aminopterin encapsulated by theliposome is in a HEPES buffered solution within the liposome.

In other embodiments, Lp-γPAMN composition is anionic or neutral. Insome embodiments, the Lp-γPAMN liposome is anionic or neutral and has adiameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175nm, or 50 nm to 150 nm, or any range therein between. In furtherembodiments, the Lp-γPAMN liposome is anionic or neutral and thecomposition has a diameter in the range of 80 nm to 120 nm, or any rangetherein between. In some embodiments, the Lp-γPAMN liposome is anionicand has a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30nm to 175 nm, or 50 nm to 150 nm, or any range therein between. Infurther embodiments, the Lp-γPAMN liposome is anionic and thecomposition has a diameter in the range of 80 nm to 120 nm, or any rangetherein between. In some embodiments, the Lp-γPAMN liposome is neutraland has a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30nm to 175 nm, or 50 nm to 150 nm, or any range therein between. In someembodiments, the anionic or neutral Lp-γPAMN composition comprises atleast 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, or more than 75%, w/w of the gamma polyglutamated AMN. In someembodiments, during the process of preparing the Lp-γPAMN, at least 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, or more than 75%, of the starting material of gamma polyglutamatedAMN is encapsulated (entrapped) in the anionic or neutral Lp-γPAMN. Insome embodiments, the anionic or neutral Lp-γPAMN composition comprisesat least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, or more than 75%, w/w of the gamma tetraglutamated AMN.In some embodiments, the anionic or neutral Lp-γPAMN compositioncomprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the gammapentaglutamated AMN. In some embodiments, the anionic or neutralLp-γPAMN composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%,35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of thegamma hexaglutamated AMN. In additional embodiments, the gammapolyglutamated aminopterin encapsulated by the liposome is in a HEPESbuffered solution within the liposome.

In additional embodiments, the liposomal gamma polyglutamatedaminopterin composition is pegylated (PLp-γPAMN).

In some embodiments, the liposomal gamma polyglutamated aminopterincomposition is non-targeted (NTLp-γPAMN). That is, the NTLp-γPAMNcomposition does not have specific affinity towards an epitope (e.g., anepitope on a surface antigen) expressed on the surface of a target cellof interest. In further embodiments, the non-targeted liposomal gammapolyglutamated aminopterin composition is pegylated (NTPLp-γPAMN).

In other embodiments, the liposomal gamma polyglutamated aminopterincomposition is targeted (TLp-γPAMN). That is, the TLp-γPAMN compositioncontains a targeting moiety that has specific affinity for an epitope(surface antigen) on a target cell of interest. In some embodiments, thetargeting moiety of the TLp-γPAMN or TPLp-γPAMN is not attached to theliposome through a covalent bond. In other embodiments, the targetingmoiety of the TLp-γPAMN or TPLp-γPAMN is attached to one or both of aPEG and the exterior of the liposome. Targeted liposomal gammapolyglutamated aminopterin compositions (TLp-γPAMN and TPLp-γPAMN)provide further improvements over the efficacy and safety profile ofaminopterin, by specifically delivering gamma polyglutamated (e.g.,γ-pentaglutamated and/or γ-hexaglutamated) aminopterin to target cellssuch as cancer cells. In some embodiments, the targeted liposomal gammapolyglutamated aminopterin composition is pegylated (TPLp-γPAMN). Insome embodiments, the targeting moiety of the TLp-γPAMN or TPLp-γPAMN isattached to one or both of a PEG and the exterior of the liposome. Insome embodiments, the targeting moiety of the TLp-γPAMN or TPLp-γPAMN isattached to the liposome through a covalent bond. Function of thetargeting moiety of the TLp-γPAMN and/or TPLp-γPAMN compositions includebut are not limited to, targeting the liposome to the target cell ofinterest in vivo or in vitro; interacting with the surface antigen forwhich the targeting moiety has specific affinity, and delivering theliposome payload (γPAMN) into the cell. Suitable targeting moieties areknown in the art and include, but are not limited to, antibodies,antigen-binding antibody fragments, scaffold proteins, polypeptides, andpeptides. In some embodiments, the targeting moiety is a polypeptide. Infurther embodiments, the targeting moiety is a polypeptide thatcomprises at least 3, 5, 10, 15, 20, 30, 40, 50, or 100, amino acidresidues.

In some embodiments, the targeting moiety of the TLp-γPAMN or TPLp-γPAMNis an antibody or an antigen-binding antibody fragment. In furtherembodiments, the targeting moiety comprises one or more of an antibody,a humanized antibody, an antigen binding fragment of an antibody, asingle chain antibody, a single-domain antibody, a bi-specific antibody,a synthetic antibody, a pegylated antibody, and a multimeric antibody.In some embodiments, the targeting moiety of the TLp-γPAMN or TPLp-γPAMNhas specific affinity for an epitope that is preferentially expressed ona target cell such as a tumor cell, compared to normal or non-tumorcells. In some embodiments, the targeting moiety has specific affinityfor an epitope on a tumor cell surface antigen that is present on atumor cell but absent or inaccessible on a non-tumor cell. In someembodiments, the targeting moiety binds an epitope of interest with anequilibrium dissociation constant (Kd) in a range of 0.5×10⁻¹⁰ to10×10⁻⁶ as determined using BIACORE analysis.

In particular embodiments, the TLp-γPAMN or TPLp-γPAMN targeting moietycomprises a polypeptide that specifically binds a folate receptor. Insome embodiments, the targeting moiety is an antibody or anantigen-binding antibody fragment. In some embodiments, the folatereceptor bound by the targeting moiety is one or more folate receptorsselected from the group consisting of: folate receptor alpha (FR-α,FOLR1), folate receptor beta (FR-β, FOLR2), and folate receptor delta(FR-δ, FOLR4). In some embodiments, the folate receptor bound by thetargeting moiety is folate receptor alpha (FR-α). In some embodiments,the folate receptor bound by the targeting moiety is folate receptorbeta (FR-β). In some embodiments, the targeting moiety specificallybinds FR-α and FR-β.

In additional embodiments, the Lp-γPAMN composition comprises one ormore of an immunostimulatory agent, a detectable marker, and amaleimide, disposed on at least one of the PEG and the exterior of theliposome. In some embodiments, the liposome γPAMN composition (e.g.,Lp-γPAMN, PLp-γPAMN, NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN, or TPLp-γPAMN)is cationic. In other embodiments, the liposome γPAMN composition (e.g.,Lp-γPAMN, PLp-γPAMN, NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN or TPLp-γPAMN)is anionic or neutral. In additional embodiments, the liposome of theliposome γPAMN composition (e.g., Lp-γPAMN, PLp-γPAMN, NTLp-γPAMN,NTPLp-γPAMN, TLp-γPAMN or TPLp-γPAMN) has a diameter in the range of 20nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, 50 nm to 150 nm, or anyrange therein between. In some embodiments, the liposome of theliposome-γPAMN composition has a diameter in the range of 30 nm to 175nm or 50 nm to 150 nm, or any range therein between. In furtherembodiments, the liposome of the liposome γPAMN composition has adiameter in the range of 80 nm to 120 nm, or any range therein between.In some embodiments, the liposome γPAMN composition is pegylated (e.g.,PLp-γPAMN, NTPLp-γPAMN, or TPLp-γPAMN). In some embodiments, theliposome γPAMN composition comprises a targeting moiety (e.g., TLp-γPAMNor TPLp-γPAMN). In further embodiments, the liposome γPAMN compositionis pegylated and targeted (e.g., TPLp-γPAMN). In some embodiments, theliposome γPAMN composition comprises gamma polyglutamated aminopterinthat contains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the liposome γPAMN composition comprises gammatetraglutamated aminopterin. In some embodiments, the liposome γPAMNcomposition comprises gamma pentaglutamated aminopterin. In otherembodiments, the liposome γPAMN composition comprises gammahexaglutamated aminopterin.

In some embodiments, the liposome compositions comprise of gammapolyglutamated aminopterin that contains 4, 5, 2-10, 4-6, or more than5, glutamyl groups and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of thegamma polyglutamated AMN. In some embodiments, the Lp-γPAMN compositioncomprises gamma polyglutamated aminopterin that contains 4, 5, 2-10,4-6, or more than 5, glutamyl groups and 1%-98.5% w/w of the gammapolyglutamated AMN. In some embodiments, the liposomes comprise gammapolyglutamated aminopterin that contains 4, 5, 2-10, 4-6, or more than5, glutamyl groups and wherein during the process of preparing theLp-γPAMN, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material ofgamma polyglutamated AMN is encapsulated (entrapped) in the Lp-γPAMN.

In some embodiments, the liposome compositions comprise of gammatetraglutamated aminopterin and at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/wof the gamma tetraglutamated AMN. In some embodiments, the Lp-γPAMNcomposition comprises gamma tetraglutamated aminopterin and 1%-98.5% w/wof the gamma tetraglutamated AMN. In some embodiments, the liposomescomprise gamma tetraglutamated aminopterin and wherein during theprocess of preparing the Lp-γPAMN, at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% ofthe starting material of gamma tetraglutamated AMN is encapsulated(entrapped) in the Lp-γPAMN.

In some embodiments, the liposome compositions comprise of gammapentaglutamated aminopterin and at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/wof the gamma pentaglutamated AMN. In some embodiments, the Lp-γPAMNcomposition comprises gamma pentaglutamated aminopterin and 1%-98.5% w/wof the gamma pentaglutamated AMN. In some embodiments, the liposomescomprise gamma pentaglutamated aminopterin and wherein during theprocess of preparing the Lp-γPAMN, at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% ofthe starting material of gamma pentaglutamated AMN is encapsulated(entrapped) in the Lp-γPAMN. In some embodiments, the liposomecompositions comprise of gamma hexaglutamated aminopterin and at least1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, or more than 75%, w/w of the gamma hexaglutamated AMN. In someembodiments, the Lp-γPAMN composition comprises gamma hexaglutamatedaminopterin and 1%-98.5% w/w of the gamma hexaglutamated AMN. In someembodiments, the liposomes comprise gamma hexaglutamated aminopterin andwherein during the process of preparing the Lp-γPAMN, at least 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, ormore than 75% of the starting material of gamma pentaglutamated AMN isencapsulated (entrapped) in the Lp-γPAMN.

Liposomal compositions comprising liposomes encapsulating γPAMN are alsoprovided. In some embodiments, the liposomal composition comprises apegylated γPAMN composition. In some embodiments, the liposomalcomposition comprises a γPAMN composition that is linked to or otherwiseassociated with a targeting moiety. In further embodiments, theliposomal composition comprises a γPAMN composition that is pegylatedand linked to or otherwise associated with a targeting moiety. In someembodiments, the liposomal composition comprises γPAMN that contains 4,5, 2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, theliposomal composition comprises gamma tetraglutamated aminopterin. Insome embodiments, the liposomal composition comprises gammapentaglutamated aminopterin. In other embodiments, the liposomalcomposition comprises gamma hexaglutamated aminopterin.

In some embodiments, the liposomal composition comprises a liposomeγPAMN (e.g., Lp-γPAMN, PLp-γPAMN, NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN,and TPLp-γPAMN). In some embodiments, the liposome γPAMN is pegylated(e.g., NTPLp-γPAMN, and TPLp-γPAMN). In some embodiments, the liposomeγPAMN comprises a targeting moiety that has a specific affinity for anepitope of antigen on the surface of a target cell of interest such as acancer cell (e.g., TLp-γPAMN or TPLp-γPAMN)). In further embodiments,the liposomal composition comprises a liposome γPAMN that is pegylatedand further comprises a targeting moiety that has a specific affinityfor an epitope of antigen on the surface of a target cell of interestsuch as a cancer cell (e.g., TPLp-γPAMN). In some embodiments, theliposomal composition comprises a liposome γPAMN that is cationic. Inother embodiments, the liposomal composition comprises a liposome γPAMNthat is anionic or neutral. In additional embodiments, the liposomalcomposition comprises a liposome γPAMN that has a diameter in the rangeof 20 nm to 500 nm, 20 nm to 200 nm, or any range therein between. Infurther embodiments, the liposome γPAMN has a diameter in the range of80 nm to 120 nm, or any range therein between.

Pharmaceutical compositions comprising gamma polyglutamated aminopterin(γPAMN) including delivery vehicles such as liposome γPAMN are alsoprovided. In some embodiments, the pharmaceutical composition comprisesa pegylated γPAMN composition. In some embodiments, the pharmaceuticalcomposition comprise a γPAMN composition that is linked to or otherwiseassociated with a targeting moiety. In further embodiments, thepharmaceutical composition comprise a γPAMN composition that ispegylated and linked to or otherwise associated with a targeting moiety.In some embodiments, the pharmaceutical composition comprises γPAMN thatcontains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the pharmaceutical composition comprises gammatetraglutamated aminopterin. In some embodiments, the pharmaceuticalcomposition comprises gamma pentaglutamated aminopterin. In otherembodiments, the pharmaceutical composition comprises gammahexaglutamated aminopterin.

In some embodiments, the pharmaceutical compositions comprise a liposomeγPAMN (e.g., Lp-γPAMN, PLp-γPAMN, NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN,and TPLp-γPAMN). In some embodiments, the liposome γPAMN composition ispegylated (e.g., NTPLp-γPAMN, and TPLp-γPAMN). In some embodiments, theliposome γPAMN comprises a targeting moiety that has a specific affinityfor an epitope of antigen on the surface of a target cell of interestsuch as a cancer cell (e.g., TLp-γPAMN or TPLp-γPAMN)). In furtherembodiments, the pharmaceutical composition comprises a liposome γPAMNcomposition that is pegylated and further comprises a targeting moietythat has a specific affinity for an epitope of antigen on the surface ofa target cell of interest such as a cancer cell (e.g., TPLp-γPAMN). Insome embodiments, the pharmaceutical composition comprises a liposomeγPAMN that is cationic. In other embodiments, the pharmaceuticalcomposition comprises a liposome γPAMN that is anionic or neutral. Inadditional embodiments, the pharmaceutical composition comprises aliposome γPAMN that has a diameter in the range of 20 nm to 500 nm or 20nm to 500 nm, or any range therein between. In further embodiments, theliposome γPAMN composition has a diameter in the range of 80 nm to 120nm, or any range therein between.

In additional embodiments, the disclosure provides a method of killing acell that comprises contacting the cell with a composition comprising agamma polyglutamated aminopterin (γPAMN) composition (e.g., a γPAMNdisclosed herein). In some embodiments, the contacted cell is amammalian cell. In further embodiments, the contacted cell is a humancell. In some embodiments, the contacted cell is a hyperproliferativecell. In further embodiments, the hyperproliferative cell is a cancercell. In further embodiments, the contacted cancer cell is a primarycell or a cell from a cell line obtained/derived from a cancer selectedfrom the group consisting of: a non-hematologic malignancy includingsuch as for example, lung cancer, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, head and neck cancer, gastric cancer,gastrointestinal cancer, colorectal cancer, esophageal cancer, cervicalcancer, liver cancer, kidney cancer, biliary duct cancer, gallbladdercancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer,central nervous system cancer, and melanoma; and a hematologicmalignancy such as for example, a leukemia, a lymphoma and other B cellmalignancies, myeloma and other plasma cell dysplasias or dyscrasias. Infurther embodiments, the contacted cancer cell is a primary cell or acell from a cell line obtained/derived from a cancer selected from thegroup consisting of: breast cancer, head and neck cancer, lung cancer,stomach cancer, osteosarcoma, Non-Hodgkin's lymphoma (NHL), acutelymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-celllymphoma) choriocarcinoma, and chorioadenoma, nonleukemic meningealcancer, soft tissue sarcoma (desmoid tumors, aggressive fibromatosis,bladder cancer, and central Nervous System (CNS) lymphoma. In yetfurther embodiments, the cancer cell is a primary cell or a cell from acell line obtained/derived from a cancer selected from colorectalcancer, lung cancer, breast cancer, head and neck cancer, and pancreaticcancer. In some embodiments, the method is performed in vivo. In otherembodiments, the method is performed in vitro. In some embodiments, theγPAMN contains 4, 5, 2-10, 4-6, or more than 5, γ-glutamyl groups. Insome embodiments the γPAMN comprises γ-glutamyl groups in the D-form. Insome embodiments, the γPAMN contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore than 10, γ-glutamyl groups in the D-form. In some embodiments theγPAMN comprises γ-glutamyl groups in the L-form. In some embodiments,the γPAMN contains 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10,γ-glutamyl groups in the L-form. In some embodiments the γPAMN comprisesγ-glutamyl groups in the L and D-form. In some embodiments the γPAMNcontains 2, 3, 4, 5, or more than 5, γ-glutamyl groups in the L-form,and 1, 2, 3, 4, 5 or more than 5, γ-glutamyl groups in the D-form. Insome embodiments, the γPAMN composition comprises gamma tetraglutamatedaminopterin. In some embodiments, the γPAMN composition comprises gammapentaglutamated aminopterin. In other embodiments, the γPAMN compositioncomprises gamma hexaglutamated aminopterin.

In additional embodiments, the disclosure provides a method of killing acell that comprises contacting the cell with a liposome containing gammapolyglutamated aminopterin (e.g., an Lp-γPAMN such as, PLp-γPAMN,NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN or TPLp-γPAMN). In some embodiments,the contacted cell is a mammalian cell. In further embodiments, thecontacted cell is a human cell. In some embodiments, the contacted cellis a hyperproliferative cell. In yet further embodiments, the contactedhyperproliferative cell is a cancer cell. In further embodiments, thecancer cell is a primary cell or a cell from a cell lineobtained/derived from a cancer selected from the group consisting of: anon-hematologic malignancy including such as for example, lung cancer,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, headand neck cancer, gastric cancer, gastrointestinal cancer, colorectalcancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer,biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, andmelanoma; and a hematologic malignancy such as for example, a leukemia,a lymphoma and other B cell malignancies, myeloma and other plasma celldysplasias or dyscrasias. In further embodiments, the contacted cancercell is a primary cell or a cell from a cell line obtained/derived froma cancer selected from the group consisting of: breast cancer, head andneck cancer, lung cancer, stomach cancer, osteosarcoma, Non-Hodgkin'slymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides(cutaneous T-cell lymphoma) choriocarcinoma, and chorioadenoma,nonleukemic meningeal cancer, soft tissue sarcoma (desmoid tumors,aggressive fibromatosis, bladder cancer, and central Nervous System(CNS) lymphoma. In some embodiments, the cancer cell is a primary cellor a cell from a cell line obtained/derived from a cancer selected fromthe group consisting of: colorectal cancer, breast cancer, ovariancancer, lung cancer, head and neck cancer, pancreatic cancer, gastriccancer, and mesothelioma. In yet further embodiments, the cancer cell isa primary cell or a cell from a cell line obtained/derived from a cancerselected from colorectal cancer, lung cancer, breast cancer, head andneck cancer, and pancreatic cancer. In some embodiments, the method isperformed in vivo. In other embodiments, the method is performed invitro. In some embodiments, the liposome contains a γPAMN containing 4,5, 2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, theliposome contains gamma tetraglutamated aminopterin. In someembodiments, the liposome contains gamma pentaglutamated aminopterin. Inother embodiments, the liposome contains gamma hexaglutamatedaminopterin.

In some embodiments, the liposome comprises a γPAMN containing 4, 5,2-10, 4-6, or more than 5, γ-glutamyl groups. In some embodiments, theliposome comprises a γPAMN comprising a γ-glutamyl group in the D-form.In some embodiments, the liposome comprises a γPAMN containing 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamyl groups in the D-form.In some embodiments, the liposome comprises a γPAMN comprisingγ-glutamyl groups in the L-form. In some embodiments, the liposomecomprises a γPAMN containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than10, γ-glutamyl groups in the L-form. In some embodiments, the liposomecomprises a γPAMN comprising γ-glutamyl groups in the L form and the Dform. In some embodiments the liposome comprises an γPAMN containing 2,3, 4, 5, or more than 5, γ-glutamyl groups in the L-form, and 1, 2, 3,4, 5 or more than 5, γ-glutamyl groups in the D-form. In someembodiments, the liposome comprises gamma pentaglutamated aminopterin.In other embodiments, the liposome comprises gamma hexaglutamatedaminopterin.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a deliveryvehicle ((e.g., an immunoconjugate or liposome) comprising gammapolyglutamated aminopterin to a subject having or at risk of havingcancer. In some embodiments, the delivery vehicle is anantibody-containing immunoconjugate (comprising. e.g., a full-length IgGantibody, a bispecific antibody, or a scFv). In some embodiments, thedelivery vehicle is a liposome (e.g., an Lp-γPAMN such as, PLp-γPAMN,NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN, or TPLp-γPAMN). In some embodiments,the administered delivery vehicle is pegylated. In some embodiments, theadministered delivery vehicle is not pegylated. In additionalembodiments, the administered delivery vehicle comprises a targetingmoiety that has a specific affinity for an epitope of antigen on thesurface of a cancer cell. In additional embodiments, the deliveryvehicle comprises a targeting moiety that has specific affinity for anepitope of a cell surface antigen selected from the group consisting of:GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin,Fibronectin Extra-domain B (ED-B), VEGFR2 (CD309), Tenascin, CollagenIV, Periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, the deliveryvehicle comprises a targeting moiety that specifically binds a cellsurface antigen(s) derived from, or determined to be expressed on, aspecific subject's cancer (tumor) such as a neoantigen. In someembodiments, the targeting moiety has specific affinity for an epitopeof a cell surface antigen(s) derived from or determined to be expressedon a specific subject's tumor such as a neoantigen. In some embodiments,the targeting moiety is an antibody or an antigen binding antibodyfragment. In some embodiments, the administered delivery vehiclecomprises γPAMN containing 4, 5, 2-10, 4-6, or more than 5, γ-glutamylgroups. In some embodiments, the delivery vehicle comprises a γPAMNcontaining 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamylgroups in the D-form. In some embodiments, the delivery vehiclecomprises a γPAMN containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than10, γ-glutamyl groups in the L-form. In some embodiments the deliveryvehicle comprises an γPAMN containing 2, 3, 4, 5, or more than 5,γ-glutamyl groups in the L-form, and 1, 2, 3, 4, 5 or more than 5,γ-glutamyl groups in the D-form. In some embodiments, the administereddelivery vehicle comprises gamma tetraglutamated aminopterin. In someembodiments, the administered delivery vehicle comprises gammapentaglutamated aminopterin. In other embodiments, the administereddelivery vehicle comprises gamma hexaglutamated aminopterin. In someembodiments, the administered delivery vehicle comprises L gammapolyglutamated aminopterin. In some embodiments, the administereddelivery vehicle comprises D gamma polyglutamated aminopterin. Infurther embodiments, the administered delivery vehicle comprises L and Dgamma polyglutamated aminopterin. In some embodiments, the cancer isselected from the group consisting of: a non-hematologic malignancyincluding such as for example, lung cancer, pancreatic cancer, breastcancer, ovarian cancer, prostate cancer, head and neck cancer, gastriccancer, gastrointestinal cancer, colorectal cancer, esophageal cancer,cervical cancer, liver cancer, kidney cancer, biliary duct cancer,gallbladder cancer, bladder cancer, sarcoma, brain cancer, centralnervous system cancer, and melanoma; and a hematologic malignancy suchas for example, a leukemia, a lymphoma and other B cell malignancies,myeloma and other plasma cell dysplasias or dyscrasias. In someembodiments, the cancer is selected from the group consisting of: breastcancer, head and neck cancer, lung cancer, stomach cancer, osteosarcoma,Non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL),mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma, andchorioadenoma, nonleukemic meningeal cancer, soft tissue sarcoma(desmoid tumors, aggressive fibromatosis, bladder cancer, and centralNervous System (CNS) lymphoma. In some embodiments, the cancer isselected from the group consisting of: colorectal cancer, breast cancer,ovarian cancer, lung cancer, head and neck cancer, pancreatic cancer,gastric cancer, and mesothelioma. In yet further embodiments, the cancercell is a primary cell or a cell from a cell line obtained/derived froma cancer selected from colorectal cancer, lung cancer, breast cancer,head and neck cancer, and pancreatic cancer.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomecomprising gamma polyglutamated aminopterin (e.g., an Lp-γPAMN such as,PLp-γPAMN, NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN, or TPLp-γPAMN) to asubject having or at risk of having cancer. In some embodiments, theliposome is pegylated. In some embodiments, the liposome is notpegylated. In additional embodiments, the liposome comprises a targetingmoiety that has a specific affinity for an epitope of antigen on thesurface of a cancer cell. In additional embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeof a cell surface antigen selected from the group consisting of: GONMB,TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin,Fibronectin Extra-domain B (ED-B), VEGFR2 (CD309), Tenascin, CollagenIV, Periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and Musk. This also includes the use of cancerstem cell targeting moieties such as those targeting CD 34, CD133 andCD44, CD138, and CD15. In some embodiments, the liposome comprises atargeting moiety that has specific affinity for an epitope of a cellsurface antigen(s) derived from or determined to be expressed on aspecific subject's tumor such as a neoantigen. In some embodiments, thetargeting moiety is an antibody or an antigen binding antibody fragment.In some embodiments, the liposome comprises γPAMN containing 4, 5, 2-10,4-6, or more than 5, glutamyl groups. In some embodiments, a liposome ofthe administered liposomal composition comprises a γPAMN containingγ-glutamyl groups in the L-form. In some embodiments, a liposome of theadministered liposomal composition comprises a γPAMN containing 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10, γ-glutamyl groups in the L-form. Insome embodiments, a liposome of the administered liposomal compositioncomprises a γPAMN containing γ-glutamyl groups in the L and D-forms. Insome embodiments, a liposome of the administered liposomal compositioncomprises an γPAMN containing 2, 3, 4, 5, or more than 5, γ-glutamylgroups in the L-form, and 1, 2, 3, 4, 5 or more than 5, γ-glutamylgroups in the D-form. In some embodiments the administered liposomalcomposition comprises tetraglutamated γPAMN. In some embodiments theadministered liposomal composition comprises pentaglutamated γPAMN. Insome embodiments the administered liposomal composition compriseshexaglutamated γPAMN. In some embodiments, the cancer is selected fromthe group consisting of: lung (e.g., non-small lung cancer), pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, head and neckcancer, gastric cancer, gastrointestinal cancer, colorectal cancer,esophageal cancer, cervical cancer, liver cancer, kidney cancer, biliaryduct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, melanoma,and a hematologic malignancy (e.g., a leukemia or lymphoma). In yetfurther embodiments, the cancer cell is a primary cell or a cell from acell line obtained/derived from a cancer selected from colorectalcancer, lung cancer, breast cancer, head and neck cancer, and pancreaticcancer.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering to a subject having or at risk ofhaving cancer, an effective amount of a liposomal composition comprisinga liposome that comprises gamma polyglutamated aminopterin and atargeting moiety that has a specific affinity for an epitope of antigenon the surface of the cancer. In some embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeof a cell surface antigen selected from the group consisting of: GONMB,TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin,Fibronectin Extra-domain B (ED-B), VEGFR2 (CD309), Tenascin, CollagenIV, Periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, the liposomecomprises a targeting moiety that a targeting moiety that has specificaffinity for an epitope of a cell surface antigen(s) derived from, ordetermined to be expressed on, a specific subject's cancer (tumor) suchas a neoantigen. In some embodiments, the targeting moiety is anantibody or an antigen binding antibody fragment. In some embodiments,the liposome comprises γPAMN containing 4, 5, 2-10, 4-6, or more than 5,γ-glutamyl groups. In some embodiments, the liposome comprises a γPAMNcontaining γ-glutamyl groups in the L-form. In some embodiments, theliposome comprises a γPAMN containing 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore than 10, γ-glutamyl groups in the L-form. In some embodiments, theliposome comprises a γPAMN containing γ-glutamyl groups in the D-form.In some embodiments, the liposome comprises a γPAMN containing 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamyl groups in the D-form.In some embodiments, the liposome comprises a gamma tetraglutamatedaminopterin. In some embodiments, the liposome comprises a gammapentaglutamated aminopterin. In some embodiments, the liposome comprisesa gamma hexaglutamated aminopterin.

In some embodiments, the administered liposomal composition comprisespegylated liposomes (e.g., TPLp-γPAMN). In some embodiments, theadministered liposomal composition comprises liposomes that are notpegylated. In some embodiments, liposomes of the administered liposomalcomposition comprise a γPAMN containing 4, 5, 2-10, 4-6, or more than 5,gamma glutamyl groups. In some embodiments, a liposome of theadministered liposomal composition comprises a γPAMN containingγ-glutamyl groups in the D-form. In some embodiments, a liposome of theadministered liposomal composition comprises a γPAMN containing 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamyl groups in the D-form.In some embodiments, a liposome of the administered liposomalcomposition comprises a γPAMN containing γ-glutamyl groups in theL-form. In some embodiments, a liposome of the administered liposomalcomposition comprises a γPAMN containing 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore than 10, γ-glutamyl groups in the L-form. In some embodiments, aliposome of the administered liposomal composition comprises a γPAMNcontaining γ-glutamyl groups in the L and D-forms. In some embodiments,a liposome of the administered liposomal composition comprises an γPAMNcontaining 2, 3, 4, 5, or more than 5, γ-glutamyl groups in the L-form,and 1, 2, 3, 4, 5 or more than 5, γ-glutamyl groups in the D-form. In insome embodiments, liposomes of the administered liposomal compositioncomprise gamma tetraglutamated aminopterin. In in some embodiments,liposomes of the administered liposomal composition comprise gammapentaglutamated aminopterin. In other embodiments, liposomes of theadministered liposomal composition comprise gamma hexaglutamatedaminopterin. In some embodiments, the liposomal composition isadministered to treat a cancer selected from the group consisting of:lung cancer (e.g., non-small cell), pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, head and neck cancer, gastric cancer,gastrointestinal cancer, colorectal cancer, esophageal cancer, cervicalcancer, liver cancer, kidney cancer, biliary duct cancer, gallbladdercancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer,central nervous system cancer, melanoma, myeloma, a leukemia and alymphoma.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomalcomposition to a subject having or at risk of having a cancer thatexpresses folate receptor on its cell surface, wherein the liposomalcomposition comprises liposomes that comprise (a) gamma polyglutamatedaminopterin (γPAMN) and (b) a targeting moiety that has specific bindingaffinity for a folate receptor. In some embodiments, the targetingmoiety has specific binding affinity for folate receptor alpha (FR-α),folate receptor beta (FR-β), and/or folate receptor delta (FR-δ). Insome embodiments, the targeting moiety has a specific binding affinityfor folate receptor alpha (FR-α), folate receptor beta (FR-β), and/orfolate receptor delta (FR-δ). In some embodiments, the targeting moietyhas a specific binding affinity for folate receptor alpha (FR-α) andfolate receptor beta (FR-β). In some embodiments, the administeredliposomal composition comprises pegylated liposomes (e.g., TPLp-γPAMN).In some embodiments, the administered liposomal composition comprisesliposomes that are not pegylated. In some embodiments, liposomes of theadministered liposomal composition comprises an γPAMN containing 4, 5,2-10, 4-6, or more than 5, γ-glutamyl groups. In some embodiments, aliposome of the administered liposomal composition comprises a γPAMNcontaining 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamylgroups in the D-form. In some embodiments, a liposome of theadministered liposomal composition comprises a γPAMN containing 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10, γ-glutamyl groups in the L-form. Insome embodiments, a liposome of the administered liposomal compositioncomprises a γPAMN containing 2, 3, 4, 5, or more than 5, γ-glutamylgroups in the L-form, and 1, 2, 3, 4, 5 or more than 5, γ-glutamylgroups in the D-form. In some embodiments, a liposome of theadministered liposomal composition comprises gamma pentaglutamatedaminopterin. In other embodiments, a liposome of the administeredliposomal composition comprises gamma hexaglutamated aminopterin. Insome embodiments, the liposomal composition is administered to treat acancer selected from the group consisting of: a non-hematologicmalignancy including such as for example, lung cancer, pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, head and neckcancer, gastric cancer, gastrointestinal cancer, colorectal cancer,esophageal cancer, cervical cancer, liver cancer, kidney cancer, biliaryduct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, andmelanoma; and a hematologic malignancy such as for example, a leukemia,a lymphoma and other B cell malignancies, myeloma and other plasma celldysplasias or dyscrasias. In some embodiments, the liposomal compositionis administered to treat a cancer is selected from the group consistingof: breast cancer, head and neck cancer, lung cancer, stomach cancer,osteosarcoma, Non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia(ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma,and chorioadenoma, nonleukemic meningeal cancer, soft tissue sarcoma(desmoid tumors, aggressive fibromatosis, bladder cancer, and centralNervous System (CNS) lymphoma. In some embodiments the liposomalcomposition is administered to treat a cancer selected from the groupconsisting of: colorectal cancer, breast cancer, ovarian cancer, lungcancer, head and neck cancer, pancreatic cancer, gastric cancer, andmesothelioma.

In additional embodiments, the disclosure provides a method for cancermaintenance therapy that comprises administering an effective amount ofa liposomal composition comprising liposomes that contain gammapolyglutamated aminopterin (Lp-γPAMN) to a subject that is undergoing orhas undergone cancer therapy. In some embodiments, the administeredliposomal composition is a PLp-γPAMN, NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMNor TPLp-γPAMN. In some embodiments, the administered liposomalcomposition comprises pegylated liposomes (e.g., PLp-γPAMN, NTPLp-γPAMN,or TPLp-γPAMN). In some embodiments, the administered liposomalcomposition comprises targeted liposomes (e.g., TLp-γPAMN orTPLp-γPAMN). In some embodiments, the administered liposomal compositioncomprises liposomes that are pegylated and comprise a targeting moiety(e.g., TPLp-γPAMN). In some embodiments, a liposome of the administeredliposomal composition comprises gamma polyglutamated aminopterin thatcontains 4, 5, 2-10, 4-6, or more than 5, γ-glutamyl groups. In someembodiments, a liposome of the administered liposomal compositioncomprises a γPAMN containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than10, γ-glutamyl groups in the D-form. In some embodiments, a liposome ofthe administered liposomal composition comprises a γPAMN containing 2,3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamyl groups in theL-form. In some embodiments, a liposome of the administered liposomalcomposition comprises a γPAMN containing 2, 3, 4, 5, or more than 5,γ-glutamyl groups in the L-form, and 1, 2, 3, 4, 5 or more than 5,γ-glutamyl groups in the D-form. In some embodiments, a liposome of theadministered liposomal composition comprises gamma tetraglutamatedaminopterin. In some embodiments, a liposome of the administeredliposomal composition comprises gamma pentaglutamated aminopterin. Inother embodiments, a liposome of the administered liposomal compositioncomprises gamma hexaglutamated aminopterin.

In additional embodiments, the disclosure provides a method for treatinga disorder of the immune system that comprises administering aneffective amount of a liposomal composition comprising liposomes thatcontain gamma polyglutamated aminopterin (e.g., Lp-γPAMN, PLp-γPAMN,NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN or TPLp-γPAMN) to a subject having orat risk of having a disorder of the immune system. In some embodiments,the liposomal composition is administered to treat an autoimmunedisease. In a further embodiment, the liposomal composition isadministered to treat rheumatoid arthritis. In another embodiment, theliposomal composition is administered to treat inflammation. In someembodiments, the administered liposomal composition comprises pegylatedliposomes (e.g., PLp-γPAMN, NTPLp-γPAMN, or TPLp-γPAMN). In someembodiments, the administered liposomal composition comprises targetedliposomes (e.g., TLp-γPAMN or TPLp-γPAMN) that contain a targetingmoiety having a specific affinity for a surface antigen on a target cellof interest (e.g., an immune cell). In further embodiments, theadministered liposomal composition comprises liposomes that arepegylated and comprise a targeting moiety (e.g., TPLp-γPAMN)). In someembodiments, a liposome of the administered liposomal compositioncomprises gamma pentaglutamated aminopterin that contains 4, 5, 2-10,4-6, or more than 5, γ-glutamyl groups. In some embodiments, a liposomeof the administered liposomal composition comprises a γPAMN containing1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamyl groups in theD-form. In some embodiments, a liposome of the administered liposomalcomposition comprises a γPAMN containing 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore than 10, γ-glutamyl groups in the L-form. In some embodiments, aliposome of the administered liposomal composition comprises a γPAMNcontaining 2, 3, 4, 5, or more than 5, γ-glutamyl groups in the L-form,and 1, 2, 3, 4, 5 or more than 5, γ-glutamyl groups in the D-form. Insome embodiments, a liposome of the administered liposomal compositioncomprise gamma tetraglutamated aminopterin. In some embodiments, aliposome of the administered liposomal composition comprise gammapentaglutamated aminopterin. In other embodiments, liposomes of theadministered liposomal composition comprise gamma hexaglutamatedaminopterin.

The disclosure also provides a method of delivering gamma polyglutamatedaminopterin to a tumor and/or cancer cell that comprises: administeringto a subject having the tumor, a composition comprising gammapolyglutamated aminopterin (L-γPAMN) and a targeting moiety that has aspecific binding affinity for an epitope on a surface antigen on thetumor cell or cancer cell. In some embodiments, the administeredtargeting moiety is associated with a delivery vehicle. In someembodiments, the delivery vehicle is an antibody or an antigen bindingfragment of an antibody. In further embodiments, the delivery vehicle isa liposome. In further embodiments, the antibody, antigen-bindingantibody fragment, or liposome is pegylated liposomes (e.g.,TPLp-γPAMN). In some embodiments, the administered composition comprisesgamma polyglutamated aminopterin that contains 4, 5, 2-10, 4-6, or morethan 5, glutamyl groups. In some embodiments, the administeredcomposition comprises gamma tetraglutamated aminopterin. In someembodiments, the administered composition comprises gammapentaglutamated aminopterin. In other embodiments, the administeredcomposition comprises gamma hexaglutamated aminopterin.

In additional embodiments, the disclosure provides a method of preparinga liposomal composition that comprises a liposomal gamma polyglutamatedaminopterin (γPAMN) composition, the method comprising: forming amixture comprising: liposomal components and γ polyglutamatedaminopterin in solution; homogenizing the mixture to form liposomes inthe solution; and processing the mixture to form liposomes containingpolyglutamated aminopterin. In some embodiments, the gammapolyglutamated aminopterin contains 4, 5, 2-10, 4-6, or more than 5,γ-glutamyl groups. In some embodiments, the γPAMN composition contains1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamyl groups in theD-form. In some embodiments, the γPAMN composition contains 2, 3, 4, 5,6, 7, 8, 9, 10, or more than 10, γ-glutamyl groups in the L-form. Insome embodiments, the γPAMN composition contains 2, 3, 4, 5, or morethan 5, γ-glutamyl groups in the L-form, and 1, 2, 3, 4, 5 or more than5, γ-glutamyl groups in the D-form. In some embodiments, the γPAMNcomposition comprises gamma pentaglutamated aminopterin. In someembodiments, the γPAMN composition comprises gamma tetraglutamatedaminopterin. In other embodiments, the γPAMN composition comprises gammahexaglutamated aminopterin.

In one embodiment, the disclosure provides a kit comprising a gammapolyglutamated aminopterin composition and/or γPAMN delivery vehiclessuch as liposomes containing γPAMN and γPAMN immunoconjugates (e.g.,ADCs) described herein.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIGS. 1A-1L show chemical formulas of aminopterin (FIG. 1A), exemplarygamma aminopterin polyglutamates, aminopterin diglutamate (FIG. 1B),aminopterin triglutamate (FIGS. 1C and 1D), aminopterin tetraglutamate(FIGS. 1E and 1F), aminopterin pentaglutamates (FIGS. 1G and 1H),aminopterin hexaglutamates (FIGS. 1I and 1J), aminopterin heptaglutamate(FIGS. 1K and 1L), aminopterin octaglutamates (FIGS. 1M and 1N), andexemplary gamma aminopterin polyglutamates (FIG. 1O).

FIG. 2 presents an example dose response relationship of free pemetrexedL-gamma hexaglutamate (gG6), liposomal pemetrexed L-gamma hexaglutamate(liposomal gG6), pemetrexed, and folate receptor alpha targetingantibody (FR1Ab) liposomal pemetrexed L-gamma hexaglutamate (liposomalgG6-FR1Ab) in the NCI H2342 non-small cell lung cancer (NSCLC),adenocarcinoma subtype depicted as the percentage of viable cells after48 hours of treatment.

FIG. 3 presents an example dose response relationship of free pemetrexedL-gamma hexaglutamate (gG6), liposomal pemetrexed L-gamma hexaglutamate(liposomal gG6), pemetrexed, and folate receptor alpha targetingantibody (FR1Ab) liposomal pemetrexed L-gamma hexaglutamate (liposomalgG6-FR1Ab) in the HT-29 (colon cancer) at 48 hours.

FIG. 4 shows the effect of free pemetrexed L-gamma hexaglutamate (hexagG6) and liposomal pemetrexed L-gamma hexaglutamate (liposomal hexagG6), on the growth of colon cancer SW260 cells following exposure of256 nM of the corresponding agent for 48 hours. The non-targeted andtargeted liposomal pemetrexed hexa gG6 are able to enter cells moreefficiently than free pemetrexed hexa gG6 to inhibit growth of the coloncancer SW260 cells.

FIG. 5 presents the relative potency of liposomal pemetrexedL-gammahexaglutamate (liposomal gG6) and its mirror image, liposomalpemetrexed gamma-D hexaglutamate (liposomal gDG6) relative to pemetrexedfollowing exposure of the cancer cell lines SW620 (CRC), HT-29 (coloncancer), H1806 (triple negative breast cancer), OAW28 (ovarian cancer),H292 (NSCLC, adenocarcinoma subtype), and H2342 (NSCLC, adenocarcinomasubtype), over 48 hours.

FIG. 6 presents the treatment effect on HCC1806 triple negative breastcancer cells following exposure of liposomal pemetrexed gamma-Lhexaglutamate (Lps Hexa gG6), liposomal pemetrexed gamma-D hexaglutamate(Lps Hexa gDG6), and to pemetrexed over 48 hours.

FIG. 7 presents the treatment effect on OAW28 ovarian cancer cellsfollowing exposure of liposomal pemetrexed gamma-L hexaglutamate (LpsHexa gG6), liposomal pemetrexed gamma-D hexaglutamate (Lps Hexa gDG6),as compared to pemetrexed over 48 hours.

FIG. 8 presents the treatment effect on H292 non-small cell lung cancercells following exposure of liposomal pemetrexed gamma-L hexaglutamate(Lps Hexa gG6), liposomal pemetrexed gamma-D hexaglutamate (Lps HexagDG6), and to pemetrexed over 48 hours.

FIG. 9 presents the treatment effect on H292 non-small cell lung cancercells following exposure of various dose levels ranging from 16 to 128nM of liposomal pemetrexed gamma-L hexaglutamate (Liposomal gG6),liposomal pemetrexed gamma-D hexaglutamate (Liposomal gDG6), andpemetrexed over 48 hours. At each of the tested dose ranges, theliposomal pemetrexed gG6 formulation is superior to inhibiting H292non-small cell lung cancer cells compared to pemetrexed.

FIG. 10 presents the treatment effect on HCC1806 triple negative breastcancer cells following exposure of various dose levels ranging from 16to 128 nM of liposomal pemetrexed gamma-L hexaglutamate (Liposomal gG6),liposomal pemetrexed gamma-D hexaglutamate (Liposomal gDG6), andpemetrexed over 48 hours. At each of the tested doses, the liposomalpemetrexed gG6 formulation is superior to pemetrexed in inhibitingHCC1806 triple negative breast cancer cells.

FIG. 11 presents the treatment effect on OAW28 ovarian cancer cells ofliposomal pemetrexed gamma-L hexaglutamate (LiposomalgG6), liposomalgamma-D hexaglutamate (LiposomalgDG6), and pemetrexed following exposureover 48 hours following exposure over a range of concentrations. At thedose of 128 nM, pemetrexed appears to more effective than the Liposomalpemetrexed gG6 liposomal formulation, whereas the liposomal formulationat the dose of 32 nM and 64 nM has a better treatment effect thanpemetrexed; at 16 nM the Liposomal pemetrexed gG6 treatment effect issimilar in to pemetrexed.

FIG. 12 shows the toxicity of liposomal pemetrexed gamma-L hexaglutamate(LiposomalgG6), liposomal pemetrexed gamma-D hexaglutamate (LiposomalgDG6), and pemetrexed on differentiating human neutrophils at 64 nM, 128nM, and 264 nM. The figure demonstrates that liposomal pemetrexed gG6 issignificantly less toxic to differentiating human neutrophils thanpemetrexed.

FIG. 13 shows the effect of liposomal pemetrexed gamma-L hexaglutamate(liposomalgG6), liposomal gamma-D hexaglutamate (liposomalgDG6), andpemetrexed on neutrophils (differentiated from CD34+ cells) followingexposure of various dose levels ranging from 16 to 128 nM of thecorresponding agent over 48 hours.

FIG. 14 shows the effect of liposomal pemetrexed gamma-L hexaglutamate(liposomalgG6), liposomal pemetrexed gamma-D hexaglutamate(liposomalgDG6), and pemetrexed on AML12 liver cells following exposureover 48 hours at 16 nM, 32 nM, and 64 nM, and 128 nM of thecorresponding agent. Strikingly, there does not appear to be anytoxicity to the AML12 liver cells following treatment with a liposomalpemetrexed gG6 at any of the liposomal agents at the dose levels tested.In contrast, pemetrexed treatment results in a reduction in the AML12liver cell counts of approximately 40% at all doses studied.

FIG. 15 shows the effect of liposomal pemetrexed gamma-L hexaglutamate(liposomalgG6), liposomal pemetrexed gamma-D hexaglutamate(liposomalgDG6), and pemetrexed on CCD841 colon epithelium cellsfollowing exposure over 48 hours at 16 nM, 32 nM, and 64 nM, and 128 nM,of the corresponding agent. At all of the concentrations tested,pemetrexed leads to approximately a ≥50% decrease in the number ofCCD841 colon epithelium cells compared to approximately a 20% or lessdecrease in cell number after treatment with each of the liposomecompositions tested.

FIG. 16 depicts the structure of polyglutamate antifolate, Cisplatin(CDDP) and two potential gG6-Cisplatin complexes. The pH dependentformation of the interstrand and/or instrastrand coordination betweenthe carboxyl groups of the polyglutamated antifolate and cisplatin islikely to disassemble into individual molecules of gG6 and cisplatinupon encountering acidic pH of lysosomes (pH 3-5) and presence ofchloride ions inside the cells.

FIG. 17 presents the effects of liposomal aG6 treatment of mice with 40mg/kg and 80 mg/kg given once weekly for 4 weeks upon the hematologicparameters: white blood cell (WBC) counts, neutrophil counts and asplatelet counts. No appreciable decrease in mean neutrophil, mean whiteblood cell or mean platelet counts was observed.

FIG. 18 presents the effects of liposomal aG6 treatment of mice with 40mg/kg and 80 mg/kg given once weekly for 4 weeks upon hemoglobin andreticulocyte indices. There is a minimal decrease in mean hemoglobinconcentrations at the higher dose level. In parallel there is a slightincrease in mean reticulocytosis indices

FIG. 19 presents the effects of liposomal aG6 treatment of mice with 40mg/kg and 80 mg/kg given once weekly for 4 weeks upon hepatic markersincluding serum aspartate transaminase (AST) and serum alaninetransaminase (ALT) along with serum albumin. There was no appreciableincreases in liver transaminases mean AST or mean ALT levels and therewas no observed change in mean albumin levels.

FIG. 20 presents the relative tumor volume of immunodeficient femaleNu/J mice (6-8 weeks old) inoculated with NCI-H292 (Non-Small Cell LungCancer) cells and administered control, pemetrexed, and Liposomal aG6intravenously at 167 mg/kg once every three weeks. As can be seen fromthese preliminary data, liposomal aG6 provides reduced tumor controlcompared to pemetrexed.

FIGS. 21A-F present the dose response relationship of liposomalpemetrexed alpha-L triglutamate (Liposomal aG3), liposomal pemetrexedalpha-L pentaglutamate (Liposomal aG5), liposomal pemetrexed alpha-Loctaglutamate (Liposomal aG7), and a combination of liposomal pemetrexedalpha-L hexaglutamate (aG6) and alpha-L dodecaglutamate (aG12)(Liposomal aG6 and aG12), over 48 hours on H2342 (NSCLC, adenocarcinomasubtype)(FIG. 21A), H292 (NSCLC, adenocarcinoma subtype)(FIG. 21B),HT-29 (colon cancer)(FIG. 21C), HCC1806 (triple negative breastcancer)(FIG. 21D), MCF7 (ER+ breast cancer)(FIG. 21E), and OAW28(ovarian cancer)(FIG. 21F). Cell viability was determined byCellTiter-Glo® (CTG) luminescent cell viability assay essentially asdescribed in Example 1. As shown in all cell lines, the potency of eachof the polyglutamated pemetrexed liposomal compositions well exceededthat of the liposomal vehicle and empty liposome controls.

DETAILED DESCRIPTION

The disclosure generally relates to gamma polyglutamated aminopterincompositions. The compositions provide advances over prior treatments ofhyperproliferative diseases such as cancer. Methods of making,delivering and using the gamma polyglutamated aminopterin compositionsare also provided. The gamma polyglutamated compositions have uses thatinclude but are not limited to treating or preventing hyperproliferativediseases such as cancer, disorders of the immune system such asinflammation and rheumatoid arthritis, and infectious disease such asHIV and malaria.

I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure pertains.

It is understood that wherever embodiments, are described herein withthe language “comprising” otherwise analogous embodiments, described interms of “containing” “consisting of” and/or “consisting essentially of”are also provided. However, when used in the claims as transitionalphrases, each should be interpreted separately and in the appropriatelegal and factual context (e.g., in claims, the transitional phrase“comprising” is considered more of an open-ended phrase while thetransitional phrases “consisting of” is more exclusive and “consistingessentially of” achieves a middle ground).

As used herein, the singular form “a”, “an”, and “the”, includes pluralreferences unless it is expressly stated or is unambiguously clear fromthe context that such is not intended.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both A and B; A or B; A (alone); and B (alone).Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C”is intended to encompass each of the following embodiments: A, B, and C;A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A(alone); B (alone); and C (alone).

Headings and subheadings are used for convenience and/or formalcompliance only, do not limit the subject technology, and are notreferred to in connection with the interpretation of the description ofthe subject technology. Features described under one heading or onesubheading of the subject disclosure may be combined, in variousembodiments, with features described under other headings orsubheadings. Further it is not necessarily the case that all featuresunder a single heading or a single subheading are used together inembodiments.

Unless indicated otherwise, the terms “aminopterin” and “AMN” are usedinterchangeably to include a salt, acid and and/or free base form ofaminopterin (e.g., aminopterin disodium). Compositions containing a AMNsalt may further contain any of a variety of cations, such as Na⁺, Mg²⁺,K⁺, NH₄ ⁺, and/or Ca²⁺. In particular embodiments, the salts arepharmaceutically acceptable salts. In additional particular embodiments,the AMN salt contains Nat Aminopterin contains one L-gamma glutamylgroup, and is therefore considered to be monoglutamated for the purposeof this disclosure.

The terms “polyglutamated-aminopterin”, “polyglutamated-AMN”, “AMN-PG”,“PAMN” and iterations thereof, are used interchangeably herein to referto a aminopterin composition that comprises at least one glutamyl groupin addition to the glutamyl group of aminopterin (i.e., AMN-PGn, whereinn≥1). Reference to the number of glutamyl groups in an γPAMN (AMN-PG)herein takes into account the glutamyl group of aminopterin. Forexample, a AMN-PG composition containing 5 glutamyl residues in additionto the glutamyl group of AMN is referred to herein as hexaglutamatedaminopterin or aminopterin hexaglutamate. Polyglutamate chains comprisean N-terminal glutamyl group and one or more C-terminal glutamyl groups.The N-terminal glutamyl group of a polyglutamate chain is not linked toanother glutamyl group via its amine group, but is linked to one or moreglutamyl group via its carboxylic acid group. In some embodiments, theN-terminal glutamyl group of a polyglutamated-aminopterin is theglutamyl group of aminopterin. The C-terminal glutamyl group or groupsof a polyglutamate chain are linked to another glutamyl group via theiramine group, but are not linked to another glutamyl group via theircarboxylic acid group.

The terms “gamma glutamyl group”, “gamma glutamyl group”, and “gammalinkage”, as they relate to the linkage of a glutamyl group, refers to aglutamyl group that contains a gamma carboxyl group linkage. The gammalinkage can be between a glutamyl group and the glutamyl group ofaminopterin, or between a glutamyl group and a second glutamyl groupthat is not present in aminopterin (e.g., a glutamyl group within apolyglutamate chain attached to aminopterin). In some embodiments, thegamma linkage is an amide bond between the gamma carboxyl group of oneglutamyl group and a second glutamyl group. In some embodiments, thegamma linkage refers to the amide bond of the glutamyl group inaminopterin. In some embodiments, the gamma linkage is an amide bondbetween the gamma carboxyl group of one glutamyl group and a secondglutamyl group. Reference to gamma linkages are inclusive of the gammalinkage of the glutamyl group in aminopterin unless it is expresslystated or is unambiguously clear from the context that such is notintended. In some embodiments, the gamma glutamyl group is in theL-form. In some embodiments, the gamma glutamyl group is in the D-form.As discussed herein, during aminopterin therapy, aminopterin enters thecell and is polyglutamated by the enzyme folylpoly-gamma-glutamatesynthetase (FPGS), which adds L glutamyl groups serially to the gammacarboxyl group of the glutamate within aminopterin L-glutamyl group ofaminopterin. Consequently, D-gamma polyglutamated aminopterincompositions are not formed within cells during aminopterin therapy.

The terms “gamma polyglutamated aminopterin”, “γ-polyglutamatedaminopterin”, “γPAMN”, “gamma polyglutamated-aminopterin”,“polyglutamated-AMN”, “γAMN-PG”, and iterations thereof, are usedinterchangeably herein to refer to a aminopterin composition thatcomprises at least one gamma glutamyl group having a gamma carboxylgroup linkage in addition to the gamma glutamyl group of aminopterin(e.g., AMN-PGn, wherein n≥1γ glutamyl group). Reference to the number ofglutamyl groups in a γPAMN (γAMN-PG) herein takes into account theglutamyl group of aminopterin. For example, a γAMN-PG compositioncontaining 5 γ-glutamyl groups in addition to the glutamyl group of AMNmay be referred to herein as gamma hexaglutamated aminopterin or gammaaminopterin hexaglutamate.

The terms “alpha glutamyl group”, “α-glutamyl group”, and “alphalinkage”, as they relate to the linkage of a glutamyl group, refers to aglutamyl group that contains an alpha carboxyl group linkage.

As use herein, the term “isolated” refers to a composition which is in aform not found in nature. Isolated gamma polyglutamated compositionsinclude those which have been purified to a degree that they are nolonger in a form in which they are found in nature. In some embodiments,a gamma polyglutamated aminopterin which is isolated is substantiallypure. Isolated compositions will be free or substantially free ofmaterial with which they are naturally associated such as other cellularcomponents such as proteins and nucleic acids with which they maypotentially be found in nature, or the environment in which they areprepared (e.g., cell culture). The gamma polyglutamated compositions maybe formulated with diluents or adjuvants and still for practicalpurposes be isolated—for example, the gamma polyglutamated compositionswill normally be mixed with pharmaceutically acceptable carriers ordiluents when used in diagnosis or therapy. In some embodiments, theisolated gamma polyglutamated compositions (e.g., gamma polyglutamatesand delivery vehicles such as liposomes containing the gammapolyglutamate contain less than 1% or less than 0.1% undesired DNA orprotein content. In some embodiments, the gamma polyglutamatecompositions (e.g., gamma polyglutamate and delivery vehicles such asliposomes containing the gamma polyglutamate) are “isolated.”

The term “targeting moiety” is used herein to refer to a molecule thatprovides an enhanced affinity for a selected target, e.g., a cell, celltype, tissue, organ, region of the body, or a compartment, e.g., acellular, tissue or organ compartment. The targeting moiety can comprisea wide variety of entities. Targeting moieties can include naturallyoccurring molecules, or recombinant or synthetic molecules. In someembodiments, the targeting moiety is an antibody, antigen-bindingantibody fragment, bispecific antibody or other antibody-based moleculeor compound. In some embodiments, the targeting moiety is an aptamer,avimer, a receptor-binding ligand, a nucleic acid, a biotin-avidinbinding pair, a peptide, protein, carbohydrate, lipid, vitamin, toxin, acomponent of a microorganism, a hormone, a receptor ligand or anyderivative thereof. Other targeting moieties are known in the art andare encompassed by the disclosure.

The terms “specific affinity”, “specifically binds”, and “enhancedaffinity”, mean that a targeting moiety such as an antibody or antigenbinding antibody fragment, reacts or associates more frequently, morerapidly, with greater duration, with greater affinity, or with somecombination of the above to the epitope, protein, or target moleculethan with alternative substances, including proteins unrelated toantigens containing the target epitope. Because of the sequence identitybetween homologous proteins in different species, specific affinity can,in several embodiments, include a binding agent that recognizes anepitope on a protein and/or target molecule in more than one species.Likewise, because of homology within certain regions of polypeptidesequences of different proteins, the term “specific affinity” or“specifically binds” can include a binding agent that recognizes anepitope that is present on more than one protein and/or target molecule.It is understood that, in certain embodiments, a targeting moiety thatspecifically binds a first target may or may not specifically bind asecond target. As such, “specific affinity” does not necessarily require(although it can include) exclusive binding, e.g., binding to an epitopeon a single target. Thus, a targeting moiety may, in certainembodiments, specifically bind an epitope that is present on more thanone target. In certain embodiments, multiple targets may be bound by thesame targeting moiety specifically binds an epitope that is present onmultiple targets.

The term “epitope” refers to that portion of an antigen capable of beingrecognized and specifically bound by a targeting moiety (i.e., bindingmoiety) such as an antibody. When the antigen is a polypeptide, epitopescan be formed both from contiguous amino acids and noncontiguous aminoacids juxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained upon protein denaturing,whereas epitopes formed by tertiary folding are typically lost uponprotein denaturing. An epitope typically includes at least 3, and moreusually, at least 5 or 8-10 amino acids in a unique spatialconformation.

Expressions like “binding affinity for a target”, “binding to a target”,“enhanced affinity”, and analogous expressions known in the art refer toa property of a targeting moiety which may be directly measured throughthe determination of the affinity constants, e.g., the amount oftargeting moiety that associates and dissociates at a given antigenconcentration. Different methods can be used to characterize themolecular interaction, such as, but not limited to, competitionanalysis, equilibrium analysis and microcalorimetric analysis, andreal-time interaction analysis based on surface plasmon resonanceinteraction (for example using a Biacore® instrument). These methods arewell-known to the skilled person and are described, for example, in Neriet al., Tibtech 14:465-470 (1996), and Jansson et al., J. Biol. Chem.272:8189-8197 (1997).

The term “delivery vehicle” refers generally to any compositions thatacts to assist, promote or facilitate entry of gamma polyglutamatedaminopterin into a cell. Such delivery vehicles are known in the art andinclude, but are not limited to, liposomes, lipospheres, polymers (e.g.,polymer-conjugates), peptides, proteins such as antibodies (e.g.,immunoconjugates, such as Antibody Drug Conjugates (ADCs) and antigenbinding antibody fragments and derivatives thereof), cellularcomponents, cyclic oligosaccharides (e.g., cyclodextrins), micelles,microparticles (e.g., microspheres), nanoparticles (e.g., lipidnanoparticles, biodegradable nanoparticles, and core-shellnanoparticles), hydrogels, lipoprotein particles, viral sequences, viralmaterial, or lipid or liposome formulations, and combinations thereof.The delivery vehicle can be linked directly or indirectly to a targetingmoiety. In some examples, the targeting moiety is selected from among amacromolecule, a protein, a peptide, a monoclonal antibody or a fattyacid lipid.

A “subject” refers to a human or vertebrate mammal including but notlimited to a dog, cat, horse, goat and primate, e.g., monkey. Thus, theinvention can also be used to treat diseases or conditions in non-humansubjects. For instance, cancer is one of the leading causes of death incompanion animals (e.g., cats and dogs). In some embodiments, of theinvention, the subject is a human. In this disclosure, the term“subject” and “patient” is used interchangeably and has the samemeaning. It is preferred generally that a maximum dose be used, that is,the highest safe dose according to sound medical judgment.

As used herein an “effective amount” refers to a dosage of an agentsufficient to provide a medically desirable result. The effective amountwill vary with the desired outcome, the particular condition beingtreated or prevented, the age and physical condition of the subjectbeing treated, the severity of the condition, the duration of thetreatment, the nature of the concurrent or combination therapy (if any),the specific route of administration and like factors within theknowledge and expertise of the health practitioner. An “effectiveamount” can be determined empirically and in a routine manner, inrelation to the stated purpose. In the case of cancer, the effectiveamount of an agent may reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and preferably stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and preferably stop) tumor metastasis; inhibit, to someextent, tumor growth; and/or relieve to some extent one or more of thesymptoms associated with the disorder. To the extent the drug mayprevent growth and/or kill existing cancer cells, it may be cytostaticand/or cytotoxic. For cancer therapy, efficacy in vivo can, for example,be measured by assessing the duration of survival, duration ofprogression free survival (PFS), the response rates (RR), duration ofresponse, and/or quality of life.

The terms “hyperproliferative disorder”, “proliferative disease”, and“proliferative disorder”, are used interchangeably herein to pertain toan unwanted or uncontrolled cellular proliferation of excessive orabnormal cells which is undesired, such as, neoplastic or hyperplasticgrowth, whether in vitro or in vivo. In some embodiments, theproliferative disease is cancer or tumor disease (including benign orcancerous) and/or any metastases, wherever the cancer, tumor and/or themetastasis is located. In some embodiments, the proliferative disease isa benign or malignant tumor. In some embodiments, the proliferativedisease is a non-cancerous disease. In some embodiments, theproliferative disease is a hyperproliferative condition such ashyperplasias, fibrosis (especially pulmonary, but also other types offibrosis, such as renal fibrosis), angiogenesis, psoriasis,atherosclerosis and smooth muscle proliferation in the blood vessels,such as stenosis or restenosis following angioplasty.

“Cancer”, “tumor”, or “malignancy”, are used as synonymous terms andrefer to any of a number of cell types or diseases that arecharacterized by uncontrolled, abnormal proliferation of cells, theability of affected cells to spread locally or through the bloodstreamand lymphatic system to other parts of the body (metastasize) and/or anyof the characteristic structural and/or molecular features known to beassociated with these cell types or diseases. “Tumor”, as used hereinrefers to all neoplastic cell growth and proliferation, whethermalignant or benign, and all pre-cancerous and cancerous cells andtissues. A “cancerous tumor”, or “malignant cell” is understood as acell having specific structural properties, lacking differentiation andbeing capable of invasion and metastasis. A cancer that can be treatedusing an γPAMN composition provided herein includes without limitation,a non-hematologic malignancy including such as for example, lung cancer,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, headand neck cancer, gastric cancer, gastrointestinal cancer, colorectalcancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer,biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, andmelanoma; and a hematologic malignancy such as for example, a leukemia,a lymphoma and other B cell malignancies, myeloma and other plasma celldysplasias or dyscrasias. In some embodiments, the cancer is selectedfrom the group consisting of: colorectal cancer, breast cancer, ovariancancer, lung cancer, head and neck cancer, pancreatic cancer, gastriccancer, and mesothelioma.

Other types of cancer and tumors that may be treated using an γPAMNcomposition are described herein or otherwise known in the art. Theterms “cancer,” “cancerous,” “cell proliferative disorder,”“proliferative disorder,” and “tumor” are not mutually exclusive asreferred to herein.

Terms such as “treating”, “treatment”, or “to treat”, refer to both (a)therapeutic measures that cure, slow down, lessen symptoms of, and/orhalt progression of a diagnosed pathologic condition or disorder and (b)prophylactic or preventative measures that prevent and/or slow thedevelopment of a targeted disease or condition. Thus, subjects in needof treatment include those already with the cancer, disorder or disease;those at risk of having the cancer or condition; and those in whom theinfection or condition is to be prevented. Subjects are identified as“having or at risk of having” cancer, an infectious disease, a disorderof the immune system, a hyperproliferative disease, or another diseaseor disorder referred to herein using well-known medical and diagnostictechniques. In certain embodiments, a subject is successfully “treated”according to the methods provided herein if the subject shows, e.g.,total, partial, or transient amelioration or elimination of a symptomassociated with the disease or condition (e.g., cancer, inflammation,and rheumatoid arthritis). In specific embodiments, the terms treating”,or “treatment”, or “to treat”, refer to the amelioration of at least onemeasurable physical parameter of a proliferative disorder, such asgrowth of a tumor, not necessarily discernible by the patient. In otherembodiments, the terms treating”, or “treatment”, or “to treat”, referto the inhibition of the progression of a proliferative disorder, eitherphysically by, e.g., stabilization of a discernible symptom,physiologically by, e.g., stabilization of a physical parameter, orboth. In other embodiments, the terms treating”, or “treatment”, or “totreat”, refer to the reduction or stabilization of tumor size, tumorcell proliferation or survival, or cancerous cell count. Treatment canbe with a γ-PAMN composition, alone or in combination with an additionaltherapeutic agent.

“Subject”, “patient”, and “animal”, are used interchangeably and referto mammals such as human patients and non-human primates, as well asexperimental animals such as rabbits, rats, and mice, and other animals.Animals include all vertebrates, e.g., mammals and non-mammals, such aschickens, amphibians, and reptiles. “Mammal” as used herein refers toany member of the class Mammalia, including, without limitation, humansand nonhuman primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingrodents such as mice, rats and guinea pigs, and other members of theclass Mammalia known in the art. In a particular embodiment, the subjectis a human.

“Treatment of a proliferative disorder” is used herein to includemaintaining or decreasing tumor size, inducing tumor regression (eitherpartial or complete), inhibiting tumor growth, and/or increasing thelife span of a subject having the proliferative disorder. In oneembodiment, the proliferative disorder is a solid tumor. Such tumorsinclude, for example, lung cancer, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, head and neck cancer, gastric cancer,gastrointestinal cancer, colorectal cancer, esophageal cancer, cervicalcancer, liver cancer, kidney cancer, biliary duct cancer, gallbladdercancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer,central nervous system cancer, and melanoma. In one embodiment, theproliferative disorder is a hematologic malignancy. Such hematologicmalignancies include for example, a leukemia, a lymphoma and other Bcell malignancies, myeloma and other plasma cell dysplasias ordyscrasias.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addison's disease, alopecia areata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, inflammation andrheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, and ulcerative colitis, among others.

The term “therapeutic agent” is used herein to refer to an agent or aderivative thereof that can interact with a hyperproliferative cell suchas a cancer cell or an immune cell, thereby reducing the proliferativestatus of the cell and/or killing the cell. Examples of therapeuticagents include, but are not limited to, chemotherapeutic agents,cytotoxic agents, platinum-based agents (e.g., cisplatin, carboplatin,oxaliplatin), taxanes (e.g., Taxol), etoposide, alkylating agents (e.g.,cyclophosphamide, ifosamide), metabolic antagonists (e.g., aminopterin(AMN), 5-fluorouracil gemcitabine, or derivatives thereof), antitumorantibiotics (e.g., mitomycin, doxorubicin), plant-derived antitumoragents (e.g., vincristine, vindesine, Taxol). Such agents may furtherinclude, but are not limited to, the anticancer agents trimetrexate,temozolomide, aminopterin, S-(4-Nitrobenzyl)-6-thioinosine (NBMPR),6-benzyguanidine (6-BG), bis-chloronitrosourea (BCNU) and camptothecin,or a therapeutic derivative of any thereof. Additional examples oftherapeutic agents that may be suitable for use in accordance with thedisclosed methods include, without limitation, anti-restenosis, pro- oranti-proliferative, anti-inflammatory, anti-neoplastic, antimitotic,anti-platelet, anticoagulant, antifibrin, antithrombin, cytostatic,antibiotic and other anti-infective agents, anti-enzymatic,anti-metabolic, angiogenic, cytoprotective, angiotensin convertingenzyme (ACE) inhibiting, angiotensin II receptor antagonizing and/orcardioprotective agents. “Therapeutic agents” also refer to salts,acids, and free based forms of the above agents.

As used herein, the term “chemotherapeutic agent” when used in relationto cancer therapy, refers to any agent that results in the death ofcancer cells or inhibits the growth or spread of cancer cells. Examplesof such chemotherapeutic agents include alkylating agents, antibiotics,antimetabolitic agents, plant-derived agents, and hormones. In someembodiments, the chemotherapeutic agent is cisplatin. In someembodiments, the chemotherapeutic agent is carboplatin. In someembodiments, the chemotherapeutic agent is oxaliplatin. In otherembodiments, the chemotherapeutic agent is gemcitabine. In otherembodiments, the chemotherapeutic agent is doxorubicin.

The term “antimetabolite” is used herein to refer to a therapeutic agentthat inhibits the utilization of a metabolite or a prodrug thereof.Examples of antimetabolites include aminopterin, aminopterin,5-fluorouracil, 5-fluorouracil prodrugs such as capecitabine,5-fluorodeoxyuridine monophosphate, cytarabine, cytarabine prodrugs suchas nelarabine, 5-azacytidine, gemcitabine, mercaptopurine, thioguanine,azathioprine, adenosine, pentostatin, erythrohydroxynonyladenine, andcladribine. Anti-metabolites useful for practicing the disclosed methodsinclude nucleoside analogs, including a purine or pyrimidine analogs. Insome embodiments, the gamma polyglutamated aminopterin compositions areused in combination with an antimetabolite selection from the groupconsisting of fluoropyrimidine 5-fluorouracil,5-fluoro-2′-deoxycytidine, cytarabine, gemcitabine, troxacitabine,decitabine, Azacytidine, pseudoisocytidine, Zebularine, Ancitabine,Fazarabine, 6-azacytidine, capecitabine, N4-octadecyl-cytarabine,elaidic acid cytarabine, fludarabine, cladribine, clofarabine,nelarabine, forodesine, and pentostatin, or a derivative thereof. In oneexample, the nucleoside analog is a substrate for a nucleoside deaminasethat is adenosine deaminase or cytidine deaminase. In some examples, thenucleoside analog is selected from among fludarabine, cytarabine,gemcitabine, decitabine and azacytidine or derivatives thereof. Incertain embodiments, the antimetabolite is 5-fluorouracil.

As used herein, a “taxane” is an anti-cancer agent that interferes withor disrupts microtubule stability, formation and/or function. Taxaneagents include paclitaxel and docetaxel as well as derivatives thereof,wherein the derivatives function against microtubules by the same modeof action as the taxane from which they are derived. In certainembodiments, the taxane is paclitaxel or docetaxel, or apharmaceutically acceptable salt, acid, or derivative of paclitaxel ordocetaxel. In certain embodiments, the taxane is paclitaxel (TAXOL®),docetaxel (TAXOTERE®), albumin-bound paclitaxel (nab-paclitaxel;ABRAXANE®), DHA-paclitaxel, or PG-paclitaxel.

The term “pharmaceutically-acceptable carrier” refers to an ingredientin a pharmaceutical formulation, other than an active ingredient, whichis nontoxic to a subject. A pharmaceutically acceptable carrierincludes, but is not limited to, a buffer, carrier, excipient,stabilizer, diluent, or preservative. Pharmaceutically-acceptablecarriers can include for example, one or more compatible solid or liquidfiller, diluents or encapsulating substances which are suitable foradministration to a human or other subject.

This disclosure generally relates gamma polyglutamated aminopterin (AMN)compositions and methods of making and using the compositions to treatdiseases including hyperproliferative diseases such as cancer, disordersof the immune system such as rheumatoid arthritis, and infectiousdisease such as HIV and malaria.

In some embodiments, the disclosure provides:

-   [1] a composition comprising a gamma polyglutamated aminopterin;-   [2] the composition of [1], wherein the gamma polyglutamated    aminopterin comprises 1-10 glutamyl groups having gamma carboxyl    group linkages;-   [3] the composition of [1] or [2], wherein the gamma polyglutamated    aminopterin contains 4, 5, 2-10, 4-6, or more than 5, glutamyl    groups having gamma carboxyl group linkages;-   [4] the composition according to any of [1]-[3], wherein the gamma    polyglutamated aminopterin is gamma tetraglutamated aminopterin;-   [5] the composition according to any of [1]-[3], wherein the gamma    polyglutamated aminopterin is gamma pentaglutamated aminopterin;-   [6] the composition according to any of [1]-[3], wherein the gamma    polyglutamated aminopterin is gamma hexaglutamated aminopterin;-   [7] the composition according to any of [1]-[6], wherein    -   (a) the gamma polyglutamated aminopterin comprises two or more        glutamyl groups in the L-form having gamma carboxyl group        linkages,    -   (b) each of the glutamyl groups of the gamma polyglutamated        aminopterin is in the L-form and has a gamma carboxyl group        linkage,    -   (c) at least one of the glutamyl groups of the gamma        polyglutamated aminopterin is in the D-form and has a gamma        carboxyl group linkage,    -   (d) each of the glutamyl groups of the gamma polyglutamated        aminopterin other than the glutamyl group of aminopterin is in        the D-form and has a gamma carboxyl group linkage, or    -   (e) the gamma polyglutamated aminopterin comprises two or more        glutamyl groups in the L-form and at least one glutamyl group in        the D-form having gamma carboxyl group linkages;-   [8] the composition according to [4], wherein (a) each of the    glutamyl groups is in the L-form and has a gamma carboxyl group    linkage or (b) each of the glutamyl groups other than the glutamyl    group of aminopterin is in the D-form and each of the glutamyl    groups has a gamma carboxyl group linkage;-   [9] the composition of [5], wherein (a) each of the glutamyl groups    is in the L-form and has a gamma carboxyl group linkage or (b) each    of the glutamyl groups other than the glutamyl group of aminopterin    is in the D-form and each of the glutamyl groups has a gamma    carboxyl group linkage;-   [10] the composition of [6], wherein (a) each of the glutamyl groups    is in the L-form and has a gamma carboxyl group linkage or (b) each    of the glutamyl groups other than the glutamyl group of aminopterin    is in the D-form and each of the glutamyl groups has a gamma    carboxyl group linkage;-   [11] the composition according to any of [1]-[10], wherein the gamma    polyglutamated aminopterin is polyglutamable by FGPS under normal    physiological conditions and/or wherein the polyglutamated AMN has a    lower uptake rate (<30%) by hepatic cells than AMN;-   [12] a liposomal composition comprising the gamma polyglutamated    aminopterin according to any of [1]-[11] (Lp-γPAMN);-   [13] the Lp-γPAMN composition according to [12], wherein the gamma    polyglutamated aminopterin comprises two or more glutamyl groups in    the L-form;-   [14] the Lp-γPAMN composition according to [12] or [13], wherein    each of the glutamyl groups of the gamma polyglutamated aminopterin    is in the L-form;-   [15] the Lp-γPAMN composition of [12] or [13], wherein at least one    of the glutamyl groups of the gamma polyglutamated aminopterin is in    the D-form;-   [16] the Lp-γPAMN composition according to any of [12]-[15], wherein    the liposome comprises a gamma polyglutamated aminopterin comprising    1-10 glutamyl groups having gamma carboxyl group linkages;-   [17] the Lp-γPAMN composition according to any of [12]-[16], wherein    the liposome comprises a gamma polyglutamated aminopterin containing    4, 5, 2-10, 4-6, or more than 5, glutamyl groups;-   [18] the Lp-γPAMN composition according to any of [12]-[17], wherein    the liposome comprises gamma tetraglutamated aminopterin;-   [19] the Lp-γPAMN composition according to any of [12]-[17], wherein    the liposome comprises gamma pentaglutamated aminopterin;-   [20] The Lp-γPAMN composition according to any of [12]-[17], wherein    the liposome comprises gamma hexaglutamated aminopterin;-   [21] the Lp-γPAMN composition according to any of [12]-[20], wherein    the liposome is not pegylated (PγLp-γPAMN);-   [22] the Lp-γPAMN composition according to any of [12]-[20], wherein    the liposome is pegylated (PγLp-γPAMN);-   [23] the Lp-γPAMN composition according to any of [12]-[22], wherein    the liposomes comprise at least 1% weight by weight (w/w) of the    gamma polyglutamated aminopterin or wherein during the process of    preparing the Lp-γPAMN, at least 1% of the starting material of    gamma polyglutamated AMN is encapsulated (entrapped) in the    Lp-γPAMN;-   [24] the Lp-γPAMN composition according to any of [12]-[24], wherein    the liposome has a diameter in the range of 20 nm to 500 nm;-   [25] the Lp-γPAMN composition according to any of [12]-[24], wherein    the liposome has a diameter in the range of 20 nm to 200 nm;-   [26] the Lp-γPAMN composition according to any of [12]-[25], wherein    the liposome has a diameter in the range of 80 nm to 120 nm;-   [27] the Lp-γPAMN composition according to any of [12]-[26], wherein    the liposome is formed from liposomal components;-   [28] the Lp-γPAMN composition according to [27], wherein the    liposomal components comprise at least one of an anionic lipid and a    neutral lipid;-   [29] the Lp-γPAMN composition according to [27] or [28], wherein the    liposomal components comprise at least one selected from the group    consisting of: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG;    cholesterol; cholesterol-PEG; and cholesterol-maleimide;-   [30] the Lp-γPAMN composition according to any of [27]-[29], wherein    the liposomal components comprise at least one selected from the    group consisting of: DSPE; DSPE-PEG; DSPE-PEG-FITC;    DSPE-PEG-maleimide; cholesterol; and HSPC;-   [31] the Lp-γPAMN composition according to any of [27]-[30], wherein    one or more liposomal components further comprises a steric    stabilizer;-   [32] the Lp-γPAMN composition according to [31], wherein the steric    stabilizer is at least one selected from the group consisting of    polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside    (GM1); poly(vinyl pyrrolidone) (PVP); poly(acrylamide) (PAA);    poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl    polyglycerol; poly[N-(2-hydroxypropyl) methacrylamide]; amphiphilic    poly-N-vinylpyrrolidones; L-amino-acid-based polymer; oligoglycerol,    copolymer containing polyethylene glycol and polypropylene oxide,    Poloxamer 188, and polyvinyl alcohol;-   [33] the Lp-γPAMN composition according to [32], wherein the steric    stabilizer is PEG and the PEG has a number average molecular weight    (Mn) of 200 to 5000 daltons;-   [34] the Lp-γPAMN composition according to any of [12]-[33], wherein    the liposome is anionic or neutral;-   [35] the Lp-γPAMN composition according to any of [12]-[33], wherein    the liposome has a zeta potential that is less than or equal to    zero;-   [36] the Lp-γPAMN composition according to any of [12]-[33], wherein    the liposome has a zeta potential that is between 0 to −150 mV;-   [37] the Lp-γPAMN composition according to any of [12]-[33], wherein    the liposome has a zeta potential that is between −30 to −50 mV;-   [38] the Lp-γPAMN composition according to any of [12]-[33], wherein    the liposome is cationic;-   [39] the Lp-γPAMN composition according to any of [12]-[38], wherein    the liposome has an interior space comprising the gamma    polyglutamated aminopterin and an aqueous pharmaceutically    acceptable carrier;-   [40] the Lp-γPAMN composition of [39], wherein the pharmaceutically    acceptable carrier comprises a tonicity agent such as dextrose,    mannitol, glycerine, potassium chloride, sodium chloride, at a    concentration of greater than 1%;-   [41] the Lp-γPAMN composition of [39], wherein the aqueous    pharmaceutically acceptable carrier is trehalose;-   [42] the Lp-γPAMN composition of [41], wherein the pharmaceutically    acceptable carrier comprises 1% to 20% trehalose;-   [43] the Lp-γPAMN composition according to any of [39]-[42], wherein    the pharmaceutically acceptable carrier comprises 1% to 50%    dextrose;-   [44] the Lp-γPAMN composition according to any of [39]-[43], wherein    the interior space of the liposome comprises 5% dextrose suspended    in an HEPES buffered solution;-   [45] the Lp-γPAMN composition according to any of [39]-[44], wherein    the pharmaceutically acceptable carrier comprises a buffer such as    HEPES Buffered Saline (HBS) or similar, at a concentration of    between 1 to 200 mM and a pH of between 2 to 8;-   [46] the Lp-γPAMN composition according to any of [39]-[45], wherein    the pharmaceutically acceptable carrier comprises a total    concentration of sodium acetate and calcium acetate of between 50 mM    to 500 mM;-   [47] the Lp-γPAMN composition according to any of [12]-[46], wherein    the interior space of the liposome has a pH of 5-8 or a pH of 6-7,    or any range therein between;-   [48] the Lp-γPAMN composition according to any of [12]-[47], wherein    the liposome comprises less than 500,000 or less than 200,000    molecules of the gamma polyglutamated aminopterin;-   [49] the Lp-γPAMN composition according to any of [12]-[48], wherein    the liposome comprises between 10 to 100,000 molecules of the gamma    polyglutamated aminopterin, or any range therein between;-   [50] the Lp-γPAMN composition according to any of [12]-[49], which    further comprises a targeting moiety and wherein the targeting    moiety has a specific affinity for a surface antigen on a target    cell of interest;-   [51] the Lp-γPAMN composition according to [50], wherein the    targeting moiety is attached to one or both of a PEG and the    exterior of the liposome, optionally wherein targeting moiety is    attached to one or both of the PEG and the exterior of the liposome    by a covalent bond;-   [52] the Lp-γPAMN composition of [50] or [51], wherein the targeting    moiety is a polypeptide;-   [53] the Lp-γPAMN composition according to any of [50]-[52], wherein    the targeting moiety is an antibody or an antigen binding fragment    of an antibody;-   [54] the Lp-γPAMN composition according to any of [50]-[53], wherein    the targeting moiety binds the surface antigen with an equilibrium    dissociation constant (Kd) in a range of 0;5×10⁻¹⁰ to 10×10⁻⁶ as    determined using BIACORE analysis;-   [55] the Lp-γPAMN composition according to any of [50]-[54], wherein    the targeting moiety specifically binds one or more folate receptors    selected from the group consisting of: folate receptor alpha (FR-α),    folate receptor beta (FR-β), and folate receptor delta (FR-δ);-   [56] the Lp-γPAMN composition according to any of [50]-[55], wherein    the targeting moiety comprises one or more selected from the group    consisting of: an antibody, a humanized antibody, an antigen binding    fragment of an antibody, a single chain antibody, a single-domain    antibody, a bi-specific antibody, a synthetic antibody, a pegylated    antibody, and a multimeric antibody;-   [57] the Lp-γPAMN composition according to any of [50]-[56], wherein    each pegylated liposome comprises from 1 to 1000 or 30-200 targeting    moieties;-   [58] the Lp-γPAMN composition according to any of [39]-[57], further    comprising one or more of an immunostimulatory agent, a detectable    marker and a maleimide, wherein the immunostimulatory agent, the    detectable marker or the maleimide is attached to said PEG or the    exterior of the liposome;-   [59] the Lp-γPAMN composition according to any of [39]-[58], wherein    the immunostimulating agent is at least one selected from the group    consisting of: a protein immunostimulating agent; a nucleic acid    immunostimulating agent; a chemical immunostimulating agent; a    hapten; and an adjuvant;-   [60] the Lp-γPAMN composition of [58] or [59], wherein the    immunostimulating agent is at least one selected from the group    consisting of: a fluorescein; a fluorescein isothiocyanate (FITC); a    DNP; a beta glucan; a beta-1,3-glucan; a beta-1,6-glucan; a resolvin    (e.g., a Resolvin D such as D_(n-6DPA) or D_(n-3DPA), a Resolvin E,    or a T series resolvin); and a Toll-like receptor (TLR) modulating    agent such as, an oxidized low-density lipoprotein (e.g., OXPAC,    PGPC), and an eritoran lipid (e.g., E5564);-   [61] the Lp-γPAMN composition according to any of [58]-[60], wherein    the immunostimulatory agent and the detectable marker is the same;-   [62] the Lp-γPAMN composition according to any of [58]-[61], further    comprising a hapten;-   [63] the Lp-γPAMN composition of [62], wherein the hapten comprises    one or more of fluorescein or Beta 1,6-glucan;-   [64] the Lp-γPAMN composition according to any of [12]-[63], which    further comprises at least one cryoprotectant selected from the    group consisting of mannitol; trehalose; sorbitol; and sucrose;-   [65] a targeted composition comprising the composition according to    any of [1]-[64];-   [66] an non-targeted composition comprising the composition    according to any of [1]-[49];-   [67] the Lp-γPAMN composition according to any of [12]-[66], which    further comprises carboplatin and/or pembroluzumab;-   [68] a pharmaceutical composition comprising the liposomal gamma    polyglutamated aminopterin composition according to any of    [12]-[67];-   [69] a pharmaceutical composition comprising gamma polyglutamated    aminopterin composition according to any of [1]-[7];-   [70] the composition of any of [1]-[69], for use in the treatment of    disease;-   [71] use of the composition of any of [1]-[70], in the manufacture    of a medicament for the treatment of disease;-   [72] a method for treating or preventing disease in a subject    needing such treatment or prevention, the method comprising    administering the composition of any of [1]-[70] to the subject;-   [73] a method for treating or preventing disease in a subject    needing such treatment or prevention, the method comprising    administering the liposomal gamma polyglutamated aminopterin    composition of any of [12]-[69] to the subject;-   [74] a method of killing a hyperproliferative cell that comprises    contacting a hyperproliferative cell with the composition of any of    [1]-[69];-   [75] a method of killing a hyperproliferative cell that comprises    contacting a hyperproliferative cell with the liposomal gamma    polyglutamated aminopterin composition of any of [12]-[69];-   [76] the method of [74] or [75], wherein the hyperproliferative cell    is a cancer cell, a mammalian cell, and/or a human cell;-   [77] a method for treating cancer that comprises administering an    effective amount of the composition of any of [1]-[69] to a subject    having or at risk of having cancer;-   [78] A method for treating cancer that comprises administering an    effective amount of the liposomal gamma polyglutamated aminopterin    composition of any of [12]-[68] to a subject having or at risk of    having cancer;-   [79] the method of [77] or [78], wherein the cancer is selected from    the group consisting of: a non-hematologic malignancy including such    as for example, lung cancer, pancreatic cancer, breast cancer,    ovarian cancer, prostate cancer, head and neck cancer, gastric    cancer, gastrointestinal cancer, colorectal cancer, esophageal    cancer, cervical cancer, liver cancer, kidney cancer, biliary duct    cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,    osteosarcoma), brain cancer, central nervous system cancer, and    melanoma; and a hematologic malignancy such as for example, a    leukemia, a lymphoma and other B cell malignancies, myeloma and    other plasma cell dyscrasias;-   [80] the method of [77] or [78], wherein the cancer is a member    selected from the group consisting of: lung cancer, breast cancer,    colon cancer, pancreatic cancer, gastric cancer, bladder cancer,    head and neck cancer, ovarian cancer, and cervical cancer;-   [81] the method of [77] or [78], wherein the cancer is a member    selected from the group consisting of: colorectal cancer, lung    cancer, breast cancer, head and neck cancer, and pancreatic cancer;-   [82] the method of [77] or [78], wherein the cancer is a sarcoma    such as osteosarcoma;-   [83] a method for treating cancer that comprises administering an    effective amount of the Lp-γPAMN composition of any of [50]-[66] to    a subject having or at risk of having a cancer cell that expresses    on its surface a folate receptor bound by the targeting moiety;-   [84] a maintenance therapy for subjects that are undergoing or have    undergone cancer therapy that comprise administering an effective    amount of the composition of any of [1]-[69] to a subject that is    undergoing or has undergone cancer therapy;-   [85] a maintenance therapy for subjects that are undergoing or have    undergone cancer therapy that comprise administering an effective    amount of the liposomal gamma polyglutamated aminopterin composition    of any of [12]-[69] to a subject that is undergoing or has undergone    cancer therapy;-   [86] a method for treating a disorder of the immune system that    comprises administering an effective amount of the composition of    any of [1]-[69] to a subject having or at risk of having a disorder    of the immune system, optionally wherein the disorder of the immune    system is selected from: inflammation (e.g., acute and chronic),    systemic inflammation, rheumatoid arthritis, inflammatory bowel    disease (IBD), Crohn disease, dermatomyositis/polymyositis, systemic    lupus erythematosus, and Takayasu, and psoriasis;-   [87] a method for treating a disorder of the immune system that    comprises administering an effective amount of the liposomal gamma    polyglutamated aminopterin composition of any of [8]-[69] to a    subject having or at risk of having a disorder of the immune system,    optionally wherein the disorder of the immune system is selected    from: inflammation (e.g., acute and chronic), systemic inflammation,    rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn    disease, dermatomyositis/polymyositis, systemic lupus erythematosus,    and Takayasu, and psoriasis;-   [88] a method for treating:    -   (a) an infectious disease that comprises administering an        effective amount of the composition according to any of [1]-[69]        to a subject having or at risk of having an infectious disease;    -   (b) an infectious disease, cardiovascular disease, metabolic        disease, or another disease, that comprises administering an        effective amount of the composition according to of any of any        of [1]-[69] to a subject having or at risk of having an        infectious disease, cardiovascular disease, or another disease,        wherein the disease is a member selected from: atherosclerosis,        cardiovascular disease (CVD), coronary artery disease,        myocardial infarction, stroke, metabolic syndrome, a gestational        trophoblastic disease, and ectopic pregnancy;    -   (c) an autoimmune disease, that comprises administering an        effective amount of the composition according to of any of any        of [1]-[69] to a subject having or at risk of having an        autoimmune disease;    -   (d) rheumatoid arthritis, that comprises administering an        effective amount of the composition according to of any of any        of [1]-[69] to a subject having or at risk of having rheumatoid        arthritis;    -   (e) an inflammatory condition that comprises administering an        effective amount of the composition according to of any of any        of [1]-[69] to a subject having or at risk of having        inflammation, optionally wherein the inflammation is acute,        chronic, and/or systemic inflammation; or    -   (f) a skin condition that comprises administering an effective        amount of the composition according to of any of claims any of        [1]-[69] to a subject having or at risk of having a skin        condition, optionally wherein the skin condition is psoriasis;-   [89] a method for treating an infectious disease that comprises    administering an effective amount of the liposomal gamma    polyglutamated aminopterin composition of any of [12]-[69] to a    subject having or at risk of having an infectious disease;-   [90] a method of delivering gamma polyglutamated aminopterin to a    tumor expressing a folate receptor on its surface, the method    comprising: administering the Lp-γPAMN composition of any of    [1]-[69] to a subject having the tumor in an amount to deliver a    therapeutically effective dose of the gamma polyglutamated    aminopterin to the tumor;-   [91] a method of preparing an gamma polyglutamated aminopterin    composition comprising the liposomal gamma polyglutamated    aminopterin composition of any of [12]-[69], the method comprising:    forming a mixture comprising: liposomal components and gamma    polyglutamated antifolate in solution; homogenizing the mixture to    form liposomes in the solution; and processing the mixture to form    liposomes containing gamma polyglutamated aminopterin;-   [92] a method of preparing an gamma polyglutamated aminopterin    composition comprising the liposomal gamma polyglutamated    aminopterin composition of any of [12]-[69], the method comprising:    forming a mixture comprising: liposomal components and gamma    polyglutamated aminopterin in solution; and processing the mixture    to form liposomes containing gamma polyglutamated aminopterin,-   [93] the method of [92], wherein the processing the mixture    comprises homogenizing the mixture to form liposomes in the    solution,-   [94] a method of preparing the composition of any of [50]-[69]    comprising the steps of:

forming a mixture comprising: liposomal components and gammapolyglutamated aminopterin in a solution; homogenizing the mixture toform liposomes in the solution; processing the mixture to form liposomesentrapping and/or encapsulating gamma polyglutamated aminopterin; andproviding a targeting moiety on a surface of the liposomes, thetargeting moiety having specific affinity for at least one of folatereceptor alpha (FR-α), folate receptor beta (FR-β) and folate receptordelta (FR-δ);

-   [95] a method of preparing the composition of any of [50]-[69],    comprising the steps of:

forming a mixture comprising: liposomal components and gammapolyglutamated aminopterin in a solution; processing the mixture to formliposomes entrapping and/or encapsulating gamma polyglutamatedaminopterin; and providing a targeting moiety on a surface of theliposomes, the targeting moiety having specific affinity for at leastone of folate receptor alpha (FR-α), folate receptor beta (FR-β) andfolate receptor delta (FR-δ);

-   [96] the method of [95], wherein the processing step comprises    homogenizing the mixture to form liposomes in the solution,-   [97] the method according to [92], wherein the processing step    includes one or more steps of: thin film hydration, extrusion,    in-line mixing, ethanol injection technique, freezing-and-thawing    technique, reverse-phase evaporation, dynamic high pressure    microfluidization, microfluidic mixing, double emulsion,    freeze-dried double emulsion, 3D printing, membrane contactor    method, and stirring; and/or-   [98] the method according to any of [95] to [97], wherein said    processing step includes one or more steps of modifying the size of    the liposomes by one or more of steps of extrusion, high-pressure    microfluidization, and/or sonication; and/or-   [99] the method of any of [91] to [98], wherein at least 1% of the    starting material of gamma polyglutamated aminopterin is    encapsulated or entrapped in the liposomes.

II. Gamma Polyglutamated Aminopterin (γPAMN)

The disclosure generally relates gamma polyglutamated aminopterin(γPAMN) compositions. The γPAMN compositions comprise at least oneglutamyl group having a gamma carboxyl group linkage. These structurallydistinct from the L-gamma polyglutamated forms of aminopterin (Lγ1PAMN)that are produced by the enzyme folylpoly-gamma-glutamate synthetase(FPGS) in cells during aminopterin therapy.

In some embodiments, the γPAMN composition contains 2-20, 2-15, 2-10,2-5, or more than 5, glutamyl groups (including the glutamyl group inaminopterin). In some embodiments, each of the glutamyl groups in theγPAMN other than the glutamyl group of aminopterin, have a gammalinkage. In some embodiments, 2 or more of the glutamyl groups in theγPAMN have a glamma linkage. In some embodiments, each of the glutamylgroups in the γPAMN is in the L-form. In some embodiments, each of theglutamyl groups in the γPAMN other than the glutamyl group ofaminopterin, is in the D-form. In some embodiments, the γPAMN comprisestwo or more glutamyl groups in the L-form and one or more glutamylgroups in the D-form.

In some embodiments, the gamma polyglutamated aminopterin isdiglutamated. That is, the gamma polyglutamated aminopterin contains 1γ-glutamyl group in addition to the glutamyl group of aminopterin(γAMN-PG₁). In some embodiments, each of the glutamyl groups of thegamma diglutamated aminopterin is in the L-form. In other embodiments,the gamma diglutamated AMN comprises a glutamyl group in the D-form.

In some embodiments, the gamma polyglutamated aminopterin istriglutamated. That is, the gamma polyglutamated aminopterin contains 2γ-glutamyl groups in addition to the glutamyl group of aminopterin(γAMN-PG₂). In some embodiments, each of the glutamyl groups of thegamma triglutamated aminopterin is in the L-form. In other embodiments,the gamma triglutamated AMN comprises a glutamyl group in the D-form. Infurther embodiments, each of the glutamyl groups of the γ-triglutamatedaminopterin other than the glutamyl group of aminopterin, is in theD-form. In additional embodiments, the γ-triglutamated AMN comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form.

In some embodiments, the gamma polyglutamated aminopterin istetraglutamated and thus contains 3 γ-glutamyl groups in addition to theγ-glutamyl group in aminopterin (γAMN-PG₃). In some embodiments, thegamma tetraglutamated AMN comprises two or more γ-glutamyl groups in theL-form. In further embodiments, each of the γ-glutamyl groups of thegamma tetraglutamated aminopterin is in the L-form. In otherembodiments, the gamma tetraglutamated AMN comprises a γ-glutamyl groupin the D-form. In some embodiments, the gamma tetraglutamated AMNcomprises 2 γ-glutamyl groups in the D-form. In some embodiments, eachof the glutamyl groups of the gamma tetraglutamated aminopterin otherthan the glutamyl group of aminopterin, is in the D-form. In additionalembodiments, the tetraglutamated AMN comprises a γ-glutamyl group in theD-form and two or more γ-glutamyl groups in the L-form.

In some embodiments, the gamma polyglutamated aminopterin ispentaglutamated (γAMN-PG₄) and contains a chain of 4 γ-glutamyl groupsattached to the glutamyl group of aminopterin. In some embodiments, thegamma pentaglutamated AMN comprises two or more glutamyl groups in theL-form. In further embodiments, each of the glutamyl groups of the gammapentaglutamated aminopterin is in the L-form. In other embodiments, thegamma pentaglutamated AMN comprises a glutamyl group in the D-form. Insome embodiments, the gamma tetraglutamated AMN comprises 2 or 3,γ-glutamyl groups in the D-form. In further embodiments, each of theγ-glutamyl groups of the gamma pentaglutamated aminopterin other thanthe glutamyl group of aminopterin, is in the D-form. In additionalembodiments, the pentaglutamated AMN comprises a γ-glutamyl group in theD-form and two or more γ-glutamyl groups in the L-form.

In some embodiments, the gamma polyglutamated aminopterin ishexaglutamated (γAMN-PG₅) and contains a chain of 5 γ-glutamyl groupsattached to the glutamyl group of aminopterin. In some embodiments, thegamma hexaglutamated AMN comprises two or more γ-glutamyl groups in theL-form. In further embodiments, each of the glutamyl groups of the gammahexaglutamated aminopterin is in the L-form. In other embodiments, thegamma hexaglutamated AMN comprises a γ-glutamyl group in the D-form. Insome embodiments, the gamma tetraglutamated AMN comprises 2, 3, 4, or 5,γ-glutamyl groups in the D-form. In further embodiments, each of theglutamyl groups of the gamma hexaglutamated aminopterin other than theglutamyl group of aminopterin, is in the D-form. In additionalembodiments, the hexaglutamated AMN comprises a γ-glutamyl group in theD-form and two or more γ-glutamyl groups in the L-form.

In some embodiments, the gamma polyglutamated aminopterin isheptaglutamated (γAMN-PG₆) and thus contains a chain of 6 γ-glutamylgroups attached to the glutamyl group of aminopterin. In someembodiments, the gamma heptaglutamated AMN comprises two or moreγ-glutamyl groups in the L-form. In further embodiments, each of theγ-glutamyl groups of the gamma heptaglutamated aminopterin is in theL-form. In other embodiments, the gamma heptaglutamated AMN comprises aγ-glutamyl group in the D-form. In some embodiments, the gammatetraglutamated AMN comprises 2, 3, 4, 5, or 6, γ-glutamyl groups in theD-form. In further embodiments, each of the γ-glutamyl groups of thegamma heptaglutamated aminopterin other than the glutamyl group ofaminopterin, is in the D-form. In additional embodiments, theheptaglutamated AMN comprises a γ-glutamyl group in the D-form and twoor more γ-glutamyl groups in the L-form.

In some embodiments, the gamma polyglutamated aminopterin isoctaglutamated (γAMN-PG₇) and thus contains a chain of 7 γ-glutamylgroups attached to the glutamyl group of aminopterin. In someembodiments, the gamma octaglutamated AMN comprises two or more glutamylgroups in the L-form. In further embodiments, each of the glutamylgroups of the gamma octaglutamated aminopterin is in the L-form. Inother embodiments, the gamma octaglutamated AMN comprises a glutamylgroup in the D-form. In some embodiments, the gamma octaglutamated AMNcomprises 2, 3, 4, 5, 6, or 7, γ-glutamyl groups in the D-form. Infurther embodiments, each of the glutamyl groups of the gammaoctaglutamated aminopterin other than the glutamyl group of aminopterin,is in the D-form. In additional embodiments, the octaglutamated AMNcomprises a glutamyl group in the D-form and two or more glutamyl groupsin the L-form.

In some embodiments, the gamma polyglutamated aminopterin isnonaglutamated (γAMN-PG₈) and contains a chain of 8 γ-glutamyl groupsattached to the glutamyl group of aminopterin. In some embodiments, thegamma nonaglutamated AMN comprises two or more glutamyl groups in theL-form. In further embodiments, each of the glutamyl groups of the gammanonaglutamated aminopterin is in the L-form. In other embodiments, thegamma nonaglutamated AMN comprises a glutamyl group in the D-form. Infurther embodiments, each of the glutamyl groups of the gammanonaglutamated aminopterin other than the glutamyl group of aminopterin,is in the D-form. In additional embodiments, the nonaglutamated AMNcomprises a γ-glutamyl group in the D-form and two or more γ-glutamylgroups in the L-form.

In some embodiments, the gamma polyglutamated aminopterin isdecaglutamated (γAMN-PG₉) and contains a chain of 9 γ-glutamyl groupsattached to the glutamyl group of aminopterin. In some embodiments, thegamma decaglutamated AMN comprises two or more glutamyl groups in theL-form. In further embodiments, each of the glutamyl groups of the gammadecaglutamated aminopterin is in the L-form. In other embodiments, thegamma decaglutamated AMN comprises a glutamyl group in the D-form. Infurther embodiments, each of the glutamyl groups of the gammadecaglutamated aminopterin other than the glutamyl group of aminopterin,is in the D-form. In additional embodiments, the decaglutamated AMNcomprises a glutamyl group in the D-form and two or more glutamyl groupsin the L-form.

In some embodiments, the gamma polyglutamated aminopterin isundecaglutamated (γAMN-PG₁₀) and contains a chain of 10 γ-glutamylgroups attached to the glutamyl group of aminopterin. In someembodiments, the gamma undecaglutamated AMN comprises two or moreglutamyl groups in the L-form. In further embodiments, each of theglutamyl groups of the gamma undecaglutamated aminopterin is in theL-form. In other embodiments, the gamma undecaglutamated AMN comprises aD glutamyl group. In further embodiments, each of the glutamyl groups ofthe gamma undecaglutamated aminopterin other than the glutamyl group ofaminopterin, is in the D-form. In additional embodiments, theundecaglutamated AMN comprises a glutamyl group in the D-form and two ormore glutamyl groups in the L-form.

In some embodiments, the gamma polyglutamated aminopterin isdodecaglutamated (γAMN-PG₁₁) and contains a chain of 11 γ-glutamylgroups attached to the glutamyl group of aminopterin. In someembodiments, the gamma dodecaglutamated AMN comprises two or moreglutamyl groups in the L-form. In further embodiments, each of theglutamyl groups of the gamma dodecaglutamated aminopterin is in theL-form. In other embodiments, the gamma dodecaglutamated AMN comprises aglutamyl group in the D-form. In further embodiments, each of theglutamyl groups of the gamma dodecaglutamated aminopterin other than theglutamyl group of aminopterin, is in the D-form. In additionalembodiments, the dodecaglutamated AMN comprises a glutamyl group in theD-form and two or more glutamyl groups in the L-form.

In some embodiments, the gamma polyglutamated aminopterin istriskaidecaglutamated (γAMN-PG₁₂) and contains a chain of 12 γ-glutamylgroups attached to the glutamyl group of aminopterin. In someembodiments, the gamma triskaidecaglutamated AMN comprises two or moreglutamyl groups in the L-form. In further embodiments, each of theglutamyl groups of the gamma triskaidecaglutamated aminopterin is in theL-form. In other embodiments, the gamma triskaidecaglutamated AMNcomprises a glutamyl group in the D-form. In further embodiments, eachof the glutamyl groups of the gamma triskaidecaglutamated aminopterinother than the glutamyl group of aminopterin, is in the D-form. Inadditional embodiments, the triskaidecaglutamated AMN comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form.

In some embodiments, the gamma polyglutamated aminopterin istetradecaglutamated (γAMN-PG₁₃) and contains a chain of 13 γ-glutamylgroups attached to the glutamyl group of aminopterin. In someembodiments, the gamma tetradecaglutamated AMN comprises two or moreglutamyl groups in the L-form. In further embodiments, each of theglutamyl groups of the gamma tetradecaglutamated aminopterin is in theL-form. In other embodiments, the gamma tetradecaglutamated AMNcomprises a glutamyl group in the D-form. In further embodiments, eachof the glutamyl groups of the gamma tetradecaglutamated aminopterinother than the glutamyl group of aminopterin, is in the D-form. Inadditional embodiments, the tetradecaglutamated AMN comprises a glutamylgroup in the D-form and two or more glutamyl groups in the L-form.

In some embodiments, the gamma polyglutamated aminopterin ispentadecaglutamated (γAMN-PG₁₄) and contains a chain of 14 γ-glutamylgroups attached to the glutamyl group of aminopterin. In someembodiments, the gamma pentadecaglutamated AMN comprises two or moreglutamyl groups in the L-form. In further embodiments, each of theglutamyl groups of the gamma pentadecaglutamated aminopterin is in theL-form. In other embodiments, the gamma pentadecaglutamated AMNcomprises a glutamyl group in the D-form. In further embodiments, eachof the glutamyl groups of the gamma pentadecaglutamated aminopterinother than the glutamyl group of aminopterin, is in the D-form. Inadditional embodiments, the pentadecaglutamated AMN comprises a glutamylgroup in the D-form and two or more glutamyl groups in the L-form.

In some embodiments, the gamma polyglutamated aminopterin ishexadecaglutamated (γAMN-PG₁₅) and contains a chain of 15 γ-glutamylgroups attached to the glutamyl group of aminopterin. In someembodiments, the gamma hexadecaglutamated AMN comprises two or moreglutamyl groups in the L-form. In further embodiments, each of theglutamyl groups of the gamma hexadecaglutamated aminopterin is in theL-form. In other embodiments, the gamma hexadecaglutamated AMN comprisesa glutamyl group in the D-form. In further embodiments, each of theglutamyl groups of the gamma hexadecaglutamated aminopterin other thanthe glutamyl group of aminopterin, is in the D-form. In additionalembodiments, the hexadecaglutamated AMN comprises a glutamyl group inthe D-form and two or more glutamyl groups in the L-form.

In other embodiments, the gamma polyglutamated aminopterin isheptadecaglutamated (γAMN-PG₁₆) and contains a chain of 16 γ-glutamylgroups attached to the glutamyl group of aminopterin. In someembodiments, the gamma heptadecaglutamated AMN comprises two or moreglutamyl groups in the L-form. In further embodiments, each of theglutamyl groups of the gamma heptadecaglutamated aminopterin is in theL-form. In other embodiments, the gamma heptadecaglutamated AMNcomprises a D glutamyl group. In further embodiments, each of theglutamyl groups of the gamma heptadecaglutamated aminopterin other thanthe glutamyl group of aminopterin, is in the D-form. In additionalembodiments, the heptadecaglutamated AMN comprises a glutamyl group inthe D-form and two or more glutamyl groups in the L-form.

In some embodiments, the gamma polyglutamated aminopterin isoctadecaglutamated (γAMN-PG₁₇) and contains a chain of 17 γ-glutamylgroups attached to the glutamyl group of aminopterin. In someembodiments, the gamma octadecaglutamated AMN comprises two or moreglutamyl groups in the L-form. In further embodiments, each of theglutamyl groups of the gamma octadecaglutamated aminopterin is in theL-form. In other embodiments, the gamma octadecaglutamated AMN comprisesa glutamyl group in the D-form. In further embodiments, each of theglutamyl groups of the gamma octadecaglutamated aminopterin other thanthe glutamyl group of aminopterin, is in the D-form. In additionalembodiments, the octadecaglutamated AMN comprises a glutamyl group inthe D-form and two or more glutamyl groups in the L-form.

In some embodiments, the gamma polyglutamated aminopterin isenneadecaglutamated (γAMN-PG₁₈) and contains a chain of 18 γ-glutamylgroups attached to the glutamyl group of aminopterin. In someembodiments, the gamma enneadecaglutamated AMN comprises two or moreglutamyl groups in the L-form. In further embodiments, each of theglutamyl groups of the gamma enneadecaglutamated aminopterin is in theL-form. In other embodiments, the gamma enneadecaglutamated AMNcomprises a D glutamyl group. In further embodiments, each of theglutamyl groups of the gamma enneadecaglutamated aminopterin other thanthe glutamyl group of aminopterin, is in the D-form. In additionalembodiments, the enneadecaglutamated AMN comprises a glutamyl group inthe D-form and two or more glutamyl groups in the L-form.

In some embodiments, the gamma polyglutamated aminopterin isicosiglutamated (γAMN-PG₁₉) and contains a chain of 19 γ-glutamyl groupsattached to the glutamyl group of aminopterin. In some embodiments, thegamma icosiglutamated AMN comprises two or more glutamyl groups in theL-form. In further embodiments, each of the glutamyl groups of the gammaicosiglutamated aminopterin is in the L-form. In other embodiments, thegamma icosiglutamated AMN comprises a glutamyl group in the D-form. Infurther embodiments, each of the glutamyl groups of the gammaicosiglutamated aminopterin other than the glutamyl group ofaminopterin, is in the D-form. In additional embodiments, theicosiglutamated AMN comprises a glutamyl group in the D-form and two ormore glutamyl groups in the L-form.

In some embodiments, the gamma polyglutamated aminopterin isicosikaihenaglutamated (γAMN-PG₂₀) and contains a chain of 20 γ-glutamylgroups attached to the glutamyl group of aminopterin. In someembodiments, the gamma icosikaihenaglutamated AMN comprises two or moreglutamyl groups in the L-form. In further embodiments, each of theglutamyl groups of the gamma icosikaihenaglutamated aminopterin is inthe L-form. In other embodiments, the gamma icosikaihenaglutamated AMNcomprises a glutamyl group in the D-form. In further embodiments, eachof the glutamyl groups of the gamma icosikaihenaglutamated aminopterinother than the glutamyl group of aminopterin, is in the D-form. Inadditional embodiments, the icosikaihenaglutamated AMN comprises aglutamyl group in the D-form and two or more glutamyl groups in theL-form.

In some embodiments, the gamma polyglutamated aminopterin contains achain of 4-7 glutamyl groups attached to aminopterin (i.e., γAMN-PGn,wherein n=4-7) and each of the 4-7 attached glutamyl groups have a gammalinkage. In some embodiments, each of the 4-7 attached glutamyl groupsis in the L-form. In other embodiments, each of the 4-7 attachedglutamyl groups is in the D-form. In other embodiments, the 4-7 attachedglutamyl groups are in the L-form and the D-form.

In one embodiment, the gamma polyglutamated aminopterin istetraglutamated and each of the 3 glutamyl groups in the polyglutamatechain attached to the aminopterin contains a gamma linkage. In someembodiments, each of the 4 glutamyl groups is in the L-form. In someembodiments, each of the glutamyl groups in the gamma tetraglutamatedaminopterin other than the glutamyl group of aminopterin, is in theD-form. In other embodiments, at least two glutamyl groups in the gammatetraglutamate aminopterin are in the L-form and at least one glutamylgroup is in the D-form.

In one embodiment, the gamma polyglutamated aminopterin ispentaglutamated and each of the 4 glutamyl groups in the polyglutamatechain attached to the aminopterin contains a gamma linkage. In someembodiments, each of the 4 glutamyl groups is in the L-form. In someembodiments, each of the glutamyl groups in the gamma pentaglutamatedaminopterin other than the glutamyl group of aminopterin, is in theD-form. In other embodiments, at least two glutamyl groups in the gammapentaglutamated aminopterin are in the L-form and at least one glutamylgroup is in the D-form.

In one embodiment, the gamma polyglutamated aminopterin ishexaglutamated. In some embodiments, each of the 5 glutamyl groups is inthe L-form. In some embodiments, each of the glutamyl groups in thegamma hexaglutamated aminopterin other than the glutamyl group ofaminopterin, is in the D-form. In other embodiments, at least twoglutamyl groups in the gamma hexaglutamated aminopterin are in theL-form and at least one glutamyl group is in the D-form.

In another embodiment, the gamma polyglutamated aminopterin isheptaglutamated. In some embodiments, each of the 6 glutamyl groups isin the L-form. In some embodiments, each of the glutamyl groups in thegamma heptaglutamated aminopterin other than the glutamyl group ofaminopterin, is in the D-form. In other embodiments, at least twoglutamyl groups in the gamma heptaglutamated aminopterin are in theL-form and at least one glutamyl group is in the D-form.

In some embodiments, the gamma polyglutamated aminopterin (γPAMN)contains a total of 1-15, 1-10, 2-15, 2-10, 3-15, 3-10, 3-6, 3-5, 4-10,4-7, or 4-6, glutamyl groups including the glutamyl group inaminopterin, or any range therein between. In some embodiments, each ofthe glutamyl groups in the γPAMN other than the glutamyl group ofaminopterin have a gamma linkage. In some embodiments, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, or 14, of the glutamyl groups in the γPAMN havea gamma linkage. In some embodiments, the γPAMN comprises γglutamylgroups in the L-form and the D-form. In some embodiments, each of theglutamyl groups in the polyglutamate structure of the polyglutamatedaminopterin is in the L-form. In some embodiments, each of the glutamylgroups in the γPAMN other than the glutamyl group of aminopterin is inthe D-form. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, or 15, of the glutamyl groups in the γPAMN is in the L-form. Inanother embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, ofthe glutamyl groups in the γPAMN is in the D-form.

In additional embodiments, the gamma polyglutamated aminopterin contains20-100, 20-75, 20-50, 20-40, 20-30, 20-25, or more than 100, gammaglutamyl groups, or any range therein between. In some embodiments, eachof the glutamyl groups of the gamma polyglutamated aminopterin is in theL-form. In other embodiments, each of the glutamyl groups of the gammapolyglutamated aminopterin other than the glutamyl group of aminopterinis in the D-form. In alternative embodiments, at least two of theglutamyl groups in the gamma polyglutamated aminopterin are in theL-form and at least one of the glutamyl groups in the gammapolyglutamated aminopterin is in the D-form

In additional embodiments, the provided compositions comprise a gammapolyglutamated aminopterin that contains 1, 2, 3, 4, 5, 6, 7, 8, 9,1-10, or 1-20, glutamyl groups that have gamma linkages. In someembodiments, the gamma polyglutamated aminopterin contains 1, 2, 3, 4,5, 6, 7, 8, 9, 1-10, or 1-20, glutamyl groups in the L-form. In someembodiments, the gamma polyglutamated aminopterin contains 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 1-10, or 1-20, glutamyl groups in the D-form. In someembodiments, the gamma polyglutamated aminopterin contains 1, 2, 3, 4,5, 6, 7, 8, 9, 1-10, or 1-20, glutamyl groups in the L-form and 0, 1, 2,3, 4, 5, 6, 7, 8, 9, or 1-10 or 1-20, glutamyl groups in the D-form.

In some embodiments, the gamma polyglutamated aminopterin compositionprovided herein is capable of adding one or more additional glutamylgroups that, is the composition is able to act as a substrate for byFPGS (folylpolyglutamate synthetase). Reagents and assays and reagentsfor determining the ability of an gamma polyglutamated aminopterincomposition to act as a substrate for FPGS (e.g., human FPGS, or ratliver FPGS) are readily available and can routinely be performed.

In some embodiments, the rate of uptake of naked gamma PPMX compositionsdisclosed herein (e.g., gamma PAMN that is not associated with adelivery vehicle) are taken up by hepatic cells at a significantlyreduced rated compared to the uptake rate of aminopterin under the samephysiological conditions. In some embodiments, the rate of hepatic celluptake of the naked gamma PAMN composition is less than 30%, 20%, 15%,or 10% compared to the rate of aminopterin. In further embodiments, therate of the efflux (transport out) of gamma PAMN compositions disclosedherein from hepatic-cells occurs at a rate that is significantly reducedcompared to aminopterin (e.g., less than 30%, 20%, 15%, or 10%) comparedto the rate of aminopterin (RT.X). n some embodiments, a gammapolyglutamated aminopterin composition provided herein is more cytotoxicto hyperproliferative cells than aminopterin. In some embodiments thehyperproliferative cells are cancer cells. In some embodiments, thehyperproliferative cells a colorectal carcinoma cells, colon cancercells, breast cancer cells, or ovarian cancer cells. In someembodiments, the cancer cells are mesothelioma cells or non-small celllung carcinoma cells. In some embodiments, cytotoxicity is measured inan in vitro assay. In some embodiments, the gamma polyglutamatedaminopterin is a hexaglutamated aminopterin.

In some embodiments, a gamma polyglutamated aminopterin compositionprovided herein has lower toxic side effects than aminopterin. In someembodiments, the gamma polyglutamated aminopterin composition providedherein is less toxic to non-hyperproliferative cells than aminopterin.In some embodiments, the gamma polyglutamated aminopterin compositionprovided herein is less toxic to neutrophils, liver cells, or to colonepithelium cells than aminopterin. In some embodiments, the neutrophilshuman neutrophils, differentiating human neutrophils, or neutrophilsdifferentiated from CD34+ cells. In some embodiments, the liver cellsare AML12 liver cells. In some embodiments, the colon epithelium cellsare CCD841 colon epithelium cells. In some embodiments, the toxicity ismeasured in an in vitro assay. In some embodiments, the gammapolyglutamated aminopterin is a hexaglutamated aminopterin.

In some embodiments, a gamma polyglutamated aminopterin aminopterincomposition provided herein has lower toxic side effects than toaminopterin. In some embodiments, a gamma polyglutamated aminopterincomposition provided herein causes fewer or less severe toxic sideeffects in an vivo assay than aminopterin. In some embodiments, the invivo assay is an in vivo murine model. In some embodiments, a gammapolyglutamated aminopterin composition provided herein causes fewer orless severe hematological or hepatic toxic side effects thanaminopterin. In some embodiments, hematological side effects areassessed by measuring mean neutrophil, mean white blood cell or meanplatelet counts. In some embodiments, hepatic toxic side effects areassessed by measuring serum aspartate transaminase (AST), serum alaninetransaminase (ALT), and/or serum albumin levels. In some embodiments,the in vivo assay comprises administering 40 mg/kg or 80 mg/kg of thegamma polyglutamated aminopterin composition once weekly for 4 weeks. Insome embodiments, the gamma polyglutamated aminopterin is ahexaglutamated aminopterin. In some embodiments, treatment with a gammapolyglutamated aminopterin composition provided herein does not inducesignificant hematological or hepatic toxic side effects in an in vivomurine model. In some embodiments, hematological side effects areassessed by measuring mean neutrophil, mean white blood cell or meanplatelet counts. In some embodiments, hepatic toxic side effects areassessed by measuring serum aspartate transaminase (AST), serum alaninetransaminase (ALT), and/or serum albumin levels. In some embodiments, agamma polyglutamated aminopterin composition provided herein does notsignificantly decrease mean neutrophil, mean white blood cell or meanplatelet counts. In some embodiments, a gamma polyglutamated aminopterincomposition provided herein does not significantly increase serumaspartate transaminase (AST) and serum alanine transaminase (ALT)levels. In some embodiments, a gamma polyglutamated aminopterincomposition provided herein does not significantly decrease serumalbumin levels. In some embodiments, the in vivo assay comprisesadministering 40 mg/kg or 80 mg/kg of the gamma polyglutamatedaminopterin composition once weekly for 4 weeks. In some embodiments,the gamma polyglutamated aminopterin is a hexaglutamated aminopterin.

In some embodiments, the gamma polyglutamated aminopterin compositionsdo not contain a fluorine atom. In some embodiments, the gammapolyglutamated aminopterin compositions do not contain a4-fluoroglutamyl group.

Gamma polyglutamated aminopterin (γPAMN) compositions and their uses arefurther disclosed in U.S. Appl. Nos. 62/374,458, 15/675,695, 15/675,701,and 62/583,432, and Intl. Appl. Nos. PCT/US2017/046666, andPCT/US2017/046667, the contents of each of which is herein incorporatedby reference in its entirety.

A. Polyglutamated Aminopterin Analogs and Derivatives

The disclosure also encompasses gamma polyglutamated aminopterinderivatives and analogs. The compositions and methods disclosed hereinare envisioned to apply to any and every known derivative or analog ofaminopterin that is polyglutamated. In some embodiments thepolyglutamated aminopterin analog or derivative composition prepared andused according to the disclosed compositions and methods is depicted inFIGS. 1I-1J. In some embodiments the analog corresponds to a modifiedform of aminopterin wherein the glutamyl group of aminopterin is notlinked to the remainder of aminopterin molecule through a gamma peptidelinkage. In some embodiments, the analog is a variant form ofaminopterin wherein the glutamyl group of aminopterin in in the D-form.In some embodiments, the polyglutamated form of aminopterin, orpolyglutamated aminopterin analog or derivative is not fluorinated.

In some embodiments, the polyglutamated aminopterin analog or derivativeencompassed by the disclosure is an indoline ring and a modifiedornithine or glutamic acid-bearing aminopterin derivative. In someembodiments, the polyglutamated aminopterin analog or derivativeencompassed by the disclosure is a member selected from the groupconsisting of: an indoline moiety-bearing aminopterin derivative, alipophilic amide aminopterin derivative, an alkyl-substituted benzenering C bearing aminopterin derivative, a polymeric platinol aminopterinderivative, a N-(L-α-aminoacyl) aminopterin derivative, a halogentatedaminopterin derivative, a 7-methyl aminopterin derivative, aN-(ac-aminoacyl) aminopterin derivative, a biotin aminopterinderivative, dichlorometho-trexate, and a lipophilic aminopterinderivative, a benzoxazine or benzothiazine moiety-bearing aminopterinderivative, and a N delta-acyl-N α-(4-amino-4-deoxypteroyl)-L-ornithinederivative,

In some embodiments, the polyglutamated aminopterin analog or derivativeencompassed by the disclosure is a member selected from the groupconsisting of: a deoxyuridylate aminopterin derivative, a10-deazaminopterin analog, a 5-deazaminopterin or a 10-deazaminopterin(10-EDAM) analog, a 5,10-dideazaminopterin aminopterin analog, a8-alkyl-7,8-dihydro analog, a L-threo-(2S,4S)-4-fluoro-glutamic acid orDL-3,3-difluoroglutamic acid-containing aminopterin analog, aaminopterin tetrahydroquinazoline analog, a D-glutamic acid, D-erythroua threo-4-fluoroglutamic acid aminopterin analog, a β γ-methanoaminopterin analog, a γ-tetrazole aminopterin analog, a or ortho isomerof aminopterin, hydroxymethylaminopterin, γ-fluoroaminopterin, agem-diphosphonate aminopterin analog, a α- or and γ-substitutedaminopterin analog, a 5-methyl-5-deaza aminopterin analog, a 8-deazaaminopterin analog, an acivicin aminopterin analog, a phosphonoglutamicacid analog, a poly (L-lysine) aminopterin conjugate, a dilysine ortrilysine aminopterin derivate,aminopterin-γ-dimyristoylphophatidylethanolamine, iodoacetyl lysineaminopterin analog, a 2,omega-diaminoalkanoid acid-containingaminopterin analog, a -methyl-5-deaza analog, a quinazoline aminopterinanalog, a pyrazine aminopterin analog, a cysteic acid or homocysteicacid aminopterin analog, a γ-tert-butyl aminopterin ester, a fluorinatedaminopterin analog, a folate aminopterin analog, a 7-hydroxyaminopterin,poly-γ-glutamyl aminopterin analog, a 3′,5′-dichloroaminopterin,diazoketone and chloromethylketone aminopterin analog, a10-propargylaminopterin or alkyl aminopterin homolog, a lectinderivative of aminopterin, a 3′,5′-dichloroaminopterin, deazaamethopterin analog, a cysteic acid and homocysteic acid aminopterinanalog, and MX068.

In additional embodiments, the gamma polyglutamated aminopterinderivative or analog has a variant polyglutamate chain. In someembodiments the polyglutamate chain contains one or more natural orsynthetic residues other than glutamate. In some embodiments thepolyglutamate chain contains one or more glutamyl groups that do notcontain an amide linkage. In other embodiments, one or more of theglutamyl groups of the polyglutamate chain is derivatized.

B. AMN-PG Synthesis

The aminopterin polyglutamate compositions provided herein may beobtained by following synthetic procedures using available reagents andsynthetic intermediates. The addition of glutamyl residues to theglutamyl residues of aminopterin can be accomplished using syntheticprocedures known in the art. In some embodiments, glutamyl residues areadded serially to the glutamyl residue of aminopterin. In additionalembodiments, polyglutamates are added to the glutamyl reside ofaminopterin using “click chemistry” methods or other bioconjugatechemistries known to those in the art. Alternatively a peptide ofglutamyl residues can be generated of the desired length and added to aprecursor of aminopterin which does not have a glutamyl residue. Thepeptide can be produced using synthetic procedures known in the art. Insome embodiments, an initial glutamyl residue is bonded to wang resinand additional glutamyl residues are added serially via solid phasepeptide synthesis using F-moc chemistry. After the final glutamylresidue is added the aminopterin precursor is coupled to the peptide andthe molecule is cleaved from the resin.

The addition of glutamyl residues to the glutamyl residues ofaminopterin can be accomplished using synthetic procedures known in theart. In some embodiments, glutamyl residues are added serially to theglutamyl residue of aminopterin. In additional embodiments,polyglutamates are added to the glutamyl reside of aminopterin using“click chemistry” methods or other bioconjugate chemistries known tothose in the art. Alternatively a peptide of glutamyl residues can begenerated of the desired length and added to a precursor of aminopterinwhich does not have a glutamyl residue. The peptide can be producedusing synthetic procedures known in the art. In some embodiments, aninitial glutamyl residue is bonded to wang resin and additional glutamylresidues are added serially via solid phase peptide synthesis usingF-moc chemistry. After the final glutamyl residue is added theaminopterin precursor is coupled to the peptide and the molecule iscleaved from the resin.

C. Aminopterin-PG Complexes

The inventors have surprising found that polyglutamated antifolates suchas aminopterin (γPAMN) are able to form complexes with othercompositions including therapeutic agents, including cytotoxic compoundssuch as platinum-based compounds. Accordingly, in some embodiments, thedisclosure provides a complex of a γPAMN (e.g., a γPAMN disclosedherein) and a therapeutic agent or a salt or acid thereof.

In some embodiments, the γPAMN/complex comprise γPAMN and a therapeuticagent. In some embodiments, the therapeutic agent is a cytotoxiccompound such as a chemotherapeutic agent. In further embodiments, theγPAMN/complex contains a platinum-based drug such as platinum-basedchemotherapeutic agent (e.g., cisplatin, carboplatin and oxaliplatin).In other embodiments, the αPAMN/complex contains a taxane-basedchemotherapeutic agent (e.g., paclitaxel and docetaxel). In otherembodiments, the γPAMN/complex contains a cyclodextrin. In furtherembodiments, the γPAMN/complex is encapsulated in a liposome

In some embodiments, the disclosure provides a composition comprising acomplex of a γPAMN and a therapeutic agent or a salt or acid thereof. Infurther embodiments, the γPAMN/therapeutic agent complex comprises oneor more γPAMN containing 2-150, 2-100, 2-75, 2-50, 2-24, 2-30, 2-20,2-19, 2-15, 2-10, or 2-5, glutamyl groups. In some embodiments, theγPAMN/therapeutic agent complex comprises one or more γPAMN containing3-10, 3-9, 3-8, or 3-7, glutamyl groups, or any range therein between.In other embodiments, the γPAMN/therapeutic agent complex comprises oneor more γPAMN containing 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5, glutamylgroups, or any range therein between. In one particular embodiment, thecomplex comprises one or more γPAMN containing 3-10 glutamyl groups. Infurther embodiments, the γPAMN/therapeutic agent complex comprises oneor more γPAMN containing 3-7 glutamyl groups. In another embodiment, theγPAMN/therapeutic agent complex comprises one or more γPAMN containing 5glutamyl groups. In another embodiment, the γPAMN/therapeutic agentcomplex comprises one or more γPAMN containing 6 glutamyl groups. Insome embodiments, the therapeutic agent is a cytotoxic compound or asalt or acid thereof. In a further embodiment, the therapeutic agent isa chemotherapeutic agent or a salt or acid thereof. In anotherembodiment, the therapeutic agent is a platinum-based drug. In anotherembodiment, the therapeutic agent is a taxane-based drug. In additionalembodiments, the molar ratio of γPAMN/therapeutic agent in the complexis in the range 1-10:1. In some embodiments, the molar ratio ofγPAMN/therapeutic agent in the complex is: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1,19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratioof γPAMN/therapeutic agent in the complex is in the range 1:1-20,1:1-10, or 1:2-8, or any range therein between. In some embodiments, themolar ratio of γPPAMN/therapeutic agent is: 1:1, 1:2, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In some embodiments, theγPAMN/therapeutic agent complex is encapsulated in a liposome (e.g., asdescribed herein or otherwise known in the art).

In an alternative embodiment, the γPAMN complex comprises γPAMN andcyclodextrin. In some embodiments, the molar ratio of γPAMN (e.g., γPAMNsalt)/cyclodextrin in the complex is in the range 1-20:1, or any rangetherein between. In some embodiments, the molar ratio ofγPAMN/cyclodextrin in the complex is in the range 1-10:1, or any rangetherein between. In further embodiments, the molar ratio ofγPAMN/cyclodextrin in the complex is in the range 2-8:1, or any rangetherein between. In some embodiments, the molar ratio ofγPAMN/cyclodextrin in the complex is: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1,8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1,20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio ofγPAMN/cyclodextrin in the complex is in the range 1:1-20, 1:1-10, or1:2-8, or any range therein between. In some embodiments, the molarratio of γPAMN/cyclodextrin is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:(21-50), or 1:>50. In some embodiments, the γPAMN/cyclodextrin complexis encapsulated in a liposome (e.g., as described herein or otherwiseknown in the art).

In some embodiments, the disclosure provides a composition comprising aγPAMN/platinum-based chemotherapeutic agent complex. In someembodiments, the platinum-based chemotherapeutic agent is selected fromthe group consisting of: cisplatin, carboplatin, and oxaliplatin, or asalt or acid thereof. In other embodiments, the γPAMN/platinum-basedchemotherapeutic agent complex comprises an analog of a cisplatin,carboplatin, oxaliplatin, or a salt or acid thereof. In someembodiments, the molar ratio of γPAMN/platinum-based agent in thecomplex is in the range 1-20:1, or any range therein between. In someembodiments, the molar ratio of γPAMN/platinum-based agent in thecomplex is in the range 1-10:1, or any range therein between. In furtherembodiments, the molar ratio of γPAMN/platinum-based agent in thecomplex is in the range 2-8:1, or any range therein between. In someembodiments, the molar ratio of γPAMN/platinum-based agent in thecomplex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1,or >50:1. In other embodiments, the molar ratio of γPAMN/platinum-basedagent in the complex is in the range 1:1-20, 1:1-10, or 1:2-8, or anyrange therein between. In some embodiments, the molar ratio ofγPAMN/platinum-based agent is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:(21-50), or 1:>50. In additional embodiments, theγPAMN//platinum-based agent complex is encapsulated in a liposome (e.g.,as described herein or otherwise known in the art).

In additional embodiments, the γPAMN/platinum-based chemotherapeuticagent complex comprises an analog of a cisplatin, carboplatin,oxaliplatin, or a salt or acid thereof. In some embodiments, the molarratio of γPAMN/platinum-based analog in the complex is in the range1-20:1, or any range therein between. In some embodiments, the molarratio of γPAMN/platinum-based analog in the complex is in the range1-10:1, or any range therein between. In further embodiments, the molarratio of γPAMN/platinum-based agent in the complex is in the range2-8:1, or any range therein between. In some embodiments, the molarratio of γPAMN/platinum-based analog in the complex is 1:1, 2:1, 3:1,4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1,17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, themolar ratio of γPAMN/platinum-based analog in the complex is in therange 1:1-20, 1:1-10, or 1:2-8, or any range therein between. In someembodiments, the molar ratio of γPAMN/platinum-based analog is: 1:1,1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14,1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additionalembodiments, the γPAMN//platinum-based analog complex is encapsulated ina liposome (e.g., as described herein or otherwise known in the art).

In further embodiments, the disclosure provides a complex containingγPAMN and cisplatin or a salt or acid thereof. In some embodiments, themolar ratio of γPAMN/cisplatin (or cisplatin salt or acid) in thecomplex is in the range 1-20:1, or any range therein between. In someembodiments, the molar ratio of γPAMN/cisplatin (or cisplatin salt oracid) in the complex is in the range 1-10:1, or any range thereinbetween. In further embodiments, the molar ratio of γPAMN/cisplatin (orcisplatin salt or acid) in the complex is in the range 2-8:1, or anyrange therein between. In some embodiments, the molar ratio ofγPAMN/cisplatin (or cisplatin salt or acid) in the complex is 1:1, 2:1,3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1,16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments,the molar ratio of γPAMN/cisplatin (or cisplatin salt or acid) in thecomplex is in the range 1:1-20, 1:1-10, or 1:2-8, or any range thereinbetween. In some embodiments, the molar ratio of γPAMN/cisplatin (orcisplatin salt or acid) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:(21-50), or 1:>50. In additional embodiments, the γPAMN//cisplatin (orcisplatin salt or acid) complex is encapsulated in a liposome (e.g., asdescribed herein or otherwise known in the art).

In another embodiment, the disclosure provides a complex containingγPAMN and carboplatin or a salt or acid thereof. In some embodiments,the molar ratio of γPAMN/carboplatin (or carboplatin salt or acid) inthe complex is in the range 1-20:1, or any range therein between. Infurther embodiments, the molar ratio of γPAMN/carboplatin (orcarboplatin salt or acid) in the complex is in the range 1-10:1, or anyrange therein between. In further embodiments, the molar ratio ofγPAMN/carboplatin (or carboplatin salt or acid) in the complex is in therange 2-8:1, or any range therein between. In some embodiments, themolar ratio of γPAMN/carboplatin (or carboplatin salt or acid) in thecomplex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1,or >50:1. In other embodiments, the molar ratio of γPAMN/carboplatin (orcarboplatin salt or acid) in the complex is in the range 1:1-20, 1:1-10,or 1:2-8, or any range therein between. In some embodiments, the molarratio of γPAMN/carboplatin (or carboplatin salt or acid) is: 1:1, 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15,1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additionalembodiments, the γPAMN/carboplatin (or carboplatin salt or acid) complexis encapsulated in a liposome (e.g., as described herein or otherwiseknown in the art).

In another embodiment, the disclosure provides a complex containingγPAMN and oxaliplatin, or a salt or acid thereof. In some embodiments,the molar ratio of γPAMN/oxaliplatin (or oxaliplatin salt or acid) inthe complex is in the range 1-20:1, or any range therein between. Infurther embodiments, the molar ratio of γPAMN/oxaliplatin (oroxaliplatin salt or acid) in the complex is in the range 1-10:1, or anyrange therein between. In further embodiments, the molar ratio ofγPAMN/oxaliplatin (or oxaliplatin salt or acid) in the complex is in therange 2-8:1, or any range therein between. In some embodiments, themolar ratio of γPAMN/oxaliplatin (or oxaliplatin salt or acid) in thecomplex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1,or >50:1. In other embodiments, the molar ratio of γPAMN/oxaliplatin (oroxaliplatin salt or acid) in the complex is in the range 1:1-20, 1:1-10,or 1:2-8, or any range therein between. In some embodiments, the molarratio of γPAMN/oxaliplatin (or oxaliplatin salt or acid) is: 1:1, 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15,1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additionalembodiments, the γPAMN/oxaliplatin (or oxaliplatin salt or acid) complexis encapsulated in a liposome (e.g., as described herein or otherwiseknown in the art).

In additional embodiments, the disclosure provides a complex comprisingγPAMN and a platinum-based chemotherapeutic agent (platinum) selectedfrom the group consisting of: nedaplatin, heptaplatin, lobaplatin,stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin,spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin,zeniplatin, platinum-triamine, traplatin, enloplatin, JM216, NK121,CI973, DWA 2114R, NDDP, and dedaplatin, or a salt or acid thereof. Inother embodiments, the γPAMN/platinum-based chemotherapeutic agentcomplex comprises an analog of nedaplatin, heptaplatin, lobaplatin,stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin,spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin,zeniplatin, platinum-triamine, traplatin, enloplatin, JM216, NK121,CI973, DWA 2114R, NDDP, or dedaplatin, or a salt or acid thereof. Insome embodiments, the molar ratio of γPAMN/platinum (or platinum salt oracid) in the complex is in the range 1-20:1, or any range thereinbetween. In further embodiments, the molar ratio of γPAMN/platinum (orplatinum salt or acid) in the complex is in the range 1-10:1, or anyrange therein between. In further embodiments, the molar ratio ofγPAMN/platinum (or platinum salt or acid) in the complex is in the range2-8:1, or any range therein between. In some embodiments, the molarratio of γPAMN/platinum (or platinum salt or acid) in the complex is1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1,14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In otherembodiments, the molar ratio of γPAMN/platinum (or platinum salt oracid) in the complex is in the range 1:1-20, 1:1-10, or 1:2-8, or anyrange therein between. In some embodiments, the molar ratio ofγPAMN/platinum (or platinum salt or acid) is: 1:1, 1:2, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments, theγPAMN/platinum (or salt or acid or analog thereof) complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In some embodiments, the disclosure provides a composition comprising aγPAMN/taxane-based chemotherapeutic agent (taxane) complex. In someembodiments, the taxane-based chemotherapeutic agent is selected fromthe group consisting of: paclitaxel (PTX), docetaxel (DTX), larotaxel(LTX), and cabazitaxel (CTX), or a salt or acid thereof. In someembodiments, the molar ratio of γPAMN/taxane-based agent in the complexis in the range 1-20:1, or any range therein between. In furtherembodiments, the molar ratio of γPAMN/taxane (or taxane salt or acid) inthe complex is in the range 1-10:1, or any range therein between. Infurther embodiments, the molar ratio of γPAMN/taxane (or taxane salt oracid) in the complex is in the range 2-8:1, or any range thereinbetween. In some embodiments, the molar ratio of γPAMN/taxane (or taxanesalt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In other embodiments, the molar ratio ofγPAMN/taxane (or taxane salt or acid) in the complex is in the range1:1-20, 1:1-10, or 1:2-8, or any range therein between. In someembodiments, the molar ratio of γγPAMN/taxane (or taxane salt or acid)is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13,1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. Inadditional embodiments, the γPAMN/taxane-based agent complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In additional embodiments, the disclosure provides a complex comprisingγPAMN and paclitaxel (PTX), or a salt or acid thereof. In otherembodiments, the γPAMN/taxane-based chemotherapeutic agent complexcomprises an analog of paclitaxel (PTX), or a salt or acid thereof. Insome embodiments, the molar ratio of γPAMN/paclitaxel (or paclitaxelsalt or acid) in the complex is in the range 1-20:1, or any rangetherein between. In further embodiments, the molar ratio ofγPAMN/paclitaxel (or paclitaxel salt or acid) in the complex is in therange 1-10:1, or any range therein between. In further embodiments, themolar ratio of γPAMN/paclitaxel (or paclitaxel salt or acid) in thecomplex is in the range 2-8:1, or any range therein between. In someembodiments, the molar ratio of γPAMN/paclitaxel (or paclitaxel salt oracid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In other embodiments, the molar ratio ofγPAMN/paclitaxel (or paclitaxel salt or acid) in the complex is in therange 1:1-20, 1:1-10, or 1:2-8, or any range therein between. In someembodiments, the molar ratio of γPAMN/paclitaxel (or paclitaxel salt oracid) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12,1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. Inadditional embodiments, the γPAMN/paclitaxel (or paclitaxel salt oracid) complex is encapsulated in a liposome (e.g., as described hereinor otherwise known in the art).

In additional embodiments, the disclosure provides a complex comprisingγPAMN and docetaxel (DTX), or a salt or acid thereof. In otherembodiments, the γPAMN/taxane-based chemotherapeutic agent complexcomprises an analog of docetaxel (DTX), or a salt or acid thereof. Insome embodiments, the molar ratio of γPAMN/docetaxel (or docetaxel saltor acid) in the complex is in the range 1-20:1, or any range thereinbetween. In some embodiments, the molar ratio of γPAMN/docetaxel (ordocetaxel salt or acid) in the complex is in the range 1-10:1, or anyrange therein between. In further embodiments, the molar ratio ofγPAMN/docetaxel (or docetaxel salt or acid) in the complex is in therange 2-8:1, or any range therein between. In some embodiments, themolar ratio of γPAMN/docetaxel (or docetaxel salt or acid) in thecomplex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1,or >50:1. In other embodiments, the molar ratio of γPAMN/docetaxel (ordocetaxel salt or acid) in the complex is in the range 1:1-20, 1:1-10,or 1:2-8, or any range therein between. In some embodiments, the molarratio of γPAMN/docetaxel (or docetaxel salt or acid) is: 1:1, 1:2, 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16,1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments,the γPAMN/docetaxel (or docetaxel salt or acid) complex is encapsulatedin a liposome (e.g., as described herein or otherwise known in the art).

In additional embodiments, the disclosure provides a complex comprisingγPAMN and larotaxel (LTX), or a salt or acid thereof. In otherembodiments, the γPAMN/taxane-based chemotherapeutic agent complexcomprises an analog of larotaxel (LTX), or a salt or acid thereof. Insome embodiments, the molar ratio of γPAMN/larotaxel (or larotaxel saltor acid) in the complex is in the range 1-20:1, or any range thereinbetween. In further embodiments, the molar ratio of γPAMN/larotaxel (orlarotaxel salt or acid) in the complex is in the range 1-10:1, or anyrange therein between. In further embodiments, the molar ratio ofγPAMN/larotaxel (or larotaxel salt or acid) in the complex is in therange 2-8:1, or any range therein between. In some embodiments, themolar ratio of γPAMN/larotaxel (or larotaxel salt or acid) in thecomplex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1,or >50:1. In other embodiments, the molar ratio of γPAMN/larotaxel (orlarotaxel salt or acid) in the complex is in the range 1:1-20, 1:1-10,or 1:2-8, or any range therein between. In some embodiments, the molarratio of γPAMN/larotaxel (or larotaxel salt or acid) is: 1:1, 1:2, 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16,1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments,the γPAMN/larotaxel (or larotaxel salt or acid) complex is encapsulatedin a liposome (e.g., as described herein or otherwise known in the art).

In additional embodiments, the disclosure provides a complex comprisingγPAMN and cabazitaxel (CTX), or a salt or acid thereof. In otherembodiments, the γPAMN/taxane-based chemotherapeutic agent complexcomprises an analog of cabazitaxel (CTX), or a salt or acid thereof. Insome embodiments, the molar ratio of γPAMN/cabazitaxel (or cabazitaxelsalt or acid) in the complex is in the range 1-20:1, or any rangetherein between. In further embodiments, the molar ratio ofγPAMN/cabazitaxel (or cabazitaxel salt or acid) in the complex is in therange 1-10:1, or any range therein between. In further embodiments, themolar ratio of γPAMN/cabazitaxel (or cabazitaxel salt or acid) in thecomplex is in the range 2-8:1, or any range therein between. In someembodiments, the molar ratio of γPAMN/cabazitaxel (or cabazitaxel saltor acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In other embodiments, the molar ratio ofγPAMN/cabazitaxel (or cabazitaxel salt or acid) in the complex is in therange 1:1-20, 1:1-10, or 1:2-8, or any range therein between. In someembodiments, the molar ratio of γPAMN/cabazitaxel (or cabazitaxel saltor acid) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11,1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or1:>50. In additional embodiments, the γPAMN/cabazitaxel (or cabazitaxelsalt or acid) complex is encapsulated in a liposome (e.g., as describedherein or otherwise known in the art).

In additional embodiments, the disclosure provides a complex comprisingγPAMN and another anti-metabolite, or a salt or acid thereof. Ananti-metabolite is a chemical with a structure that is similar to ametabolite required for normal biochemical reactions, yet differentenough to interfere with one or more normal functions of cells, such ascell division. In some embodiments, the disclosure provides a complexcomprising γPAMN and aminopterin (AMN), or a salt or acid thereof. Insome embodiments, the disclosure provides a complex comprising γPAMN andan anti-metabolite selected from the group consisting of, gemcitabine,fluorouracil, capecitabine, an antifolate (e.g., aminopterin,aminopterin), tegafur, cytosine arabinoside, thioguanine, 5-azacytidine,6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabinephosphate, and cladribine, as well as pharmaceutically acceptable saltor acids, acids, or derivatives of any of these. In some embodiments,the molar ratio of γPAMN/anti-metabolite (or anti-metabolite salt oracid) in the complex is in the range 1-20:1, or any range thereinbetween. In further embodiments, the molar ratio ofγPAMN/anti-metabolite (or anti-metabolite salt or acid) in the complexis in the range 1-10:1, or any range therein between. In furtherembodiments, the molar ratio of γPAMN/anti-metabolite (oranti-metabolite salt or acid) in the complex is in the range 2-8:1, orany range therein between. In some embodiments, the molar ratio ofγPAMN/anti-metabolite (or anti-metabolite salt or acid) in the complexis 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1,14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50)1, or >50:1. In otherembodiments, the molar ratio of γPAMN/anti-metabolite (oranti-metabolite salt or acid) in the complex is in the range 1:1-20,1:1-10, or 1:2-8, or any range therein between. In some embodiments, themolar ratio of γPAMN/anti-metabolite (or anti-metabolite salt or acid)is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13,1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. Inadditional embodiments, the γPAMN/anti-metabolite (or anti-metabolitesalt or acid) complex is encapsulated in a liposome (e.g., as describedherein or otherwise known in the art).

In additional embodiments, the disclosure provides a complex of γPAMN(e.g., an γPAMN disclosed herein) and a cyclodextrin. Cyclodextrins(CDs) are groups of cyclic oligosaccharides which have been shown toimprove physicochemical properties of many drugs through formation ofcomplexes. CDs are cyclic oligosaccharides composed of several D-glucoseunits linked by α-(1,4) bonds. This cyclic configuration provides ahydrophobic internal cavity and gives the CDs a truncated cone shape.Many hydroxyl groups are situated on the edges of the ring which makethe CDs both lipophilic and soluble in water. As a result, CDs are ableto form complexes with a wide variety of hydrophobic agents, and thuschange the physical-chemical properties of these complexed agents.

The terms “cyclodextrin” or “CD” unless otherwise specified herein,refer generally to a parent or derivatized cyclic oligosaccharidecontaining a variable number of (α-1,4)-linked D-glucopyranoside unitsthat is able to form a complex with a aminopterin-PG. Each cyclodextringlucopyranoside subunit has secondary hydroxyl groups at the 2 and 3positions and a primary hydroxyl group at the 6-position. The terms“parent”, “underivatized”, or “inert”, cyclodextrin refer to acyclodextrin containing D-glucopyranoside units having the basic formulaC₆H₁₂O₆ and a glucose structure without any additional chemicalsubstitutions (e.g., α-cyclodextrin consisting of 6 D-glucopyranosideunits, a β-cyclodextrin cyclodextrin consisting of 7 D-glucopyranosideunits, and a γ-cyclodextrin cyclodextrin consisting of 8D-glucopyranoside units). The physical and chemical properties of aparent cyclodextrin can be modified by derivatizing the hydroxyl groupswith other functional groups. Any substance located within thecyclodextrin internal phase is said to be “complexed” with thecyclodextrin, or to have formed a complex (inclusion complex) with thecyclodextrin.

As used herein, there are no particular limitations on the cyclodextrincomponent of the γPAMN/cyclodextrin complexes so long as thecyclodextrins can form complexes with the γPAMN. In particularembodiments, the cyclodextrins have been derivatized to bear ionizable(e.g., weakly basic and/or weakly acidic) functional groups tofacilitate complex formation with γPAMN and/or liposome encapsulation.

Modifications of the hydroxyl groups of cyclodextrins, such as thosefacing away from the cyclodextrin interior phase, with ionizablechemical groups is known to facilitate the loading of cyclodextrins andtherapeutic agents complexed with the cyclodextrins. In someembodiments, the cyclodextrin of the γPAMN/cyclodextrin complex has atleast 2, 3, 4, 5, 6, 6, 7, 8, 9, or 10, hydroxyl group substituted withan ionizable chemical group. The term “charged cyclodextrin” refers to acyclodextrin having one or more of its hydroxyl groups substituted witha charged moiety. Such a moiety can itself be a charged group or it cancomprise an organic moiety (e.g., a C₁-C₆ alkyl or C₁-C₆ alkyl ethermoiety) substituted with one or more charged moieties.

In some embodiments, the “ionizable” or “charged” moieties of a CDderivative are weakly ionizable. Weakly ionizable moieties are thosethat are either weakly basic or weakly acidic. Weakly basic functionalgroups (W) have a pKa of between about 6.0-9.0, 6.5-8.5, 7.0-8.0,7.5-8.0, and any range in between inclusive according to CH3-W.Similarly, weakly acidic functional groups (X) have a log dissociationconstant (pKa) of between about 3.0-7.0, 4.0-6.5, 4.5-6.5, 5.0-6.0,5.0-5.5, and any range in between inclusive according to CH3-X.Representative anionic moieties include, without limitation,carboxylate, carboxymethyl, succinyl, sulfonyl, phosphate, sulfoalkylether, sulphate carbonate, thiocarbonate, dithiocarbonate, phosphate,phosphonate, sulfonate, nitrate, and borate groups. Representativecationic moieties include, without limitation, amino, guanidine, andquarternary ammonium groups.

In another embodiment, the derivatized cyclodextrin is a “polyanion” or“polycation.” A polyanion is a derivatized cyclodextrin having more thanone negatively charged group resulting in net a negative ionic charge ofmore than two units. A polycation is a derivatized cyclodextrin havingmore than one positively charged group resulting in net positive ioniccharger of more than two units.

In another embodiment, the derivatized cyclodextrin is a “chargeableamphiphile.” By “chargeable” is meant that the amphiphile has a pK inthe range pH 4 to pH 8 or 8.5. A chargeable amphiphile may therefore bea weak acid or base. By “amphoteric” herein is meant a derivatizedcyclodextrin having a ionizable groups of both anionic and cationiccharacter wherein: (a) at least one, and optionally both, of the cationand anionic amphiphiles is chargeable, having at least one charged groupwith a pK between 4 and 8 to 8.5, (b) the cationic charge prevails at pH4, and (c) the anionic charge prevails at pH 8 to 8.5.

In some embodiments, the “ionizable” or “charged” derivatizedcyclodextrin as a whole, whether polyionic, amphiphilic, or otherwise,are weakly ionizable (e.g., have a pKai of between about 4.0-8.5,4.5-8.0, 5.0-7.5, 5.5-7.0, 6.0-6.5, and any range in between inclusive).

Any one, some, or all hydroxyl groups of any one, some or allα-D-glucopyranoside units of a cyclodextrin can be modified to anionizable chemical group as described herein. Since each cyclodextrinhydroxyl group differs in chemical reactivity, reaction with a modifyingmoiety can produce an amorphous mixture of positional and opticalisomers. Alternatively, certain chemistry can allow for pre-modifiedα-D-glucopyranoside units to be reacted to form uniform products.

The aggregate substitution that occurs for cyclodextrin derivatives in amixture is described by a term referred to as the degree ofsubstitution. For example, a 6-ethylenediamino-β-cyclodextrin with adegree of substitution of seven would be composed of a distribution ofisomers of 6-ethylenediamino-β-cyclodextrin in which the average numberof ethylenediamino groups per 6-ethylenediamino-β-cyclodextrin moleculeis seven. The degree of substitution for a cyclodextrin derivativemixture can routinely be determined using mass spectrometry or nuclearmagnetic resonance spectroscopy.

In one embodiment, at least one hydroxyl moieties facing away from thecyclodextrin interior is substituted with an ionizable chemical group.For example, the C2, C3, C6, C2 and C3, C2 and C6, C3 and C6, and allthree of C2-C3-C6 hydroxyls of at least one γ-D-glucopyranoside unit aresubstituted with an ionizable chemical group. Any such combination ofhydroxyls can similarly be combined with at least two, three, four,five, six, seven, eight, nine, ten, eleven, up to all of thealpha-D-glucopyranoside units in the modified cyclodextrin as well as incombination with any degree of substitution described herein. One suchderivative is a sulfoalkyl ether cyclodextrin (SAE-CD). Sulfobutyl etherderivatives of beta cyclodextrin (SBE-(β-CD) have been demonstrated tohave significantly improved aqueous solubility compared to the parentcyclodextrin.

Additional cyclodextrin derivatives that may be complexed withtherapeutic agents in the disclosed liposome compositions includesugammadex or Org-25969, in which the 6-hydroxy groups on γ-CD have beenreplaced by carboxythio acetate ether linkages, and hydroxybutenyl-β-CD.Alternative forms of cyclodextrin include: 2,6-Di-β-methyl-β-CD (DIMEB),2-hydroxylpropyl-3-cyclodextrin (HP-β-CD), randomlymethylated-β-cyclodextrin (RAMEB), sulfobutyl ether β-cyclodextrin(SBE-β-CD), and sulfobutylether-γ-cyclodextrin (SBEγCD), sulfobutylatedbeta-cyclodextrin sodium salt, (2-Hydroxypropyl)-gamma-cyclodextrin,(2-Hydroxypropyl)-beta-cyclodextrin, (2-Hydroxypropyl)-γ-cyclodextrin,2,6-di-O-methyl)-beta-cyclodextrin (DIMEB-50 Heptakis),2,3,6-tri-O-methyl)-beta-cyclodextrin (TRIMEB Heptakis),methyl-beta-cyclodextrin, octakis (6-deoxy-6-iodo)-γ-cyclodexrin, and,octakis (6-deoxy-6-bromo)-gamma-cyclodexrin.

In some embodiments, the cyclodextrin(s) has a high solubility in waterin order to facilitate entrapment of a larger amount of the cyclodextrinin the liposome internal phase. In some embodiments, the watersolubility of the cyclodextrin is at least 10 mg/mL, 20 mg/mL, 30 mg/mL,40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL orhigher. In some embodiments, the water solubility of the cyclodextrin(s)is within a range of 10-150 mg/mL, 20-100 mg/mL 20-75 mg/mL, and anyrange in between inclusive.

In some embodiments, a large association constant between thecyclodextrin and the γPAMN and/or other therapeutic agent complexed withcyclodextrin is preferable and can be obtained by selecting the numberof glucose units in the cyclodextrin based on the size of thetherapeutic agent (see, for example, Albers et al., Crit. Rev. Therap.Drug Carrier Syst. 12:311-337 (1995); Stella et al., Toxicol. Pathol.36:30-42 (2008). When the association constant depends on pH, thecyclodextrin can be selected such that the association constant becomeslarge at the pH of the liposome internal phase. As a result, thesolubility (nominal solubility) of the therapeutic agent in the presenceof cyclodextrin can be further improved. In some embodiments, theassociation constant of the cyclodextrin with the therapeutic agent is100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, or higher. In someembodiments, the association constant of the cyclodextrin with thetherapeutic agent is in the range 100-1, 200, 200-1,000, 300-750, andany range therein between.

In some embodiments, the cyclodextrin of the γPAMN/cyclodextrin complexand/or cyclodextrin/therapeutic agent complex is underivatized.

In some embodiments, the cyclodextrin of the γPAMN/cyclodextrin complexand/or cyclodextrin/therapeutic agent complex is derivatized. In furtherembodiments, the cyclodextrin derivative of the complex has thestructure of Formula I:

wherein: n is 4, 5, or 6;

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ are each, independently,—H, a straight chain or branched C₁-C₈-alkylene group, or an optionallysubstituted straight-chain or branched C₁-C₆ group, wherein at least oneof R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branchedC₁-C₈-alkylene (e.g., C₁-C₈-(alkylene)-SO₃ ⁻ group);

In some embodiments, the cyclodextrin derivative of theγPAMN/cyclodextrin complex and/or cyclodextrin/therapeutic agent complexhas the structure of formula II:

wherein: n is 4, 5, or 6;

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ are each, independently,—O— or a -β-(C₂-C₆ alkylene)-SO₃— group; wherein at least one of R₁ andR₂ is independently a -β-(C₂-C₆ alkylene)-SO₃ ⁻ group; and S₁, S₂, S₃,S₄, S₅, S₆, S₇, S₈, and S₉ are each, independently, a pharmaceuticallyacceptable cation. In further embodiments, the pharmaceuticallyacceptable cation is selected from: an alkali metal such as Li⁺, Na⁺, orK⁺; an alkaline earth metal such as Ca⁺², or Mg⁺² and ammonium ions andamine cations such as the cations of (C₁-C₆)-alkylamines, piperidine,pyrazine, (C₁-C₆)-alkanolamine and (C₄-C₈)-cycloalkanolamine. In someembodiments, at least one of R₁ and R₂ is independently a —O—(C₂-C₆alkylene)-SO3-group that is a -β-(CH₂)_(m)SO3-group, wherein m is 2 to6, preferably 2 to 4, (e.g., —O—CH2CH2CH2SO3- or -β-CH2CH2CH2CH2SO₃—);and S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ are each, independently, H ora pharmaceutically cation which includes for example, alkali metals(e.g., Li⁺, Na⁺, K⁺) alkaline earth metals (e.g., Ca⁺², Mg⁺²), ammoniumions and amine cations such as the cations of (C1-C6)-alkylamines,piperidine, pyrazine, (C₁-C₆)-alkanol-amine and(C₄-C₈)-cycloalkanolamine:

In some embodiments, a cyclodextrin derivative of the γPAMN/cyclodextrincomplex and/or cyclodextrin/therapeutic agent complex is a cyclodextrindisclosed in U.S. Pat. Nos. 6,133,248, 5,874,418, 6,046,177, 5,376,645,5,134,127, 7,034,013, 6,869,939; and Intl. Appl. Publ. No. WO02005/117911, the contents each of which is herein incorporated byreference in its priority.

In some embodiments, the cyclodextrin derivative of theγPAMN/cyclodextrin complex and/or cyclodextrin/therapeutic agent complexis a sulfoalkyl ether cyclodextrin. In some embodiments, thecyclodextrin derivative of complex is a sulfobutyl ether-3-cyclodextrinsuch as CAPTISOL® (CyDex Pharma. Inc., Lenexa, Kans. Methods forpreparing sulfobutyl ether-3-cyclodextrin and other sulfoalkyl ethercyclodextrins are known in the art.

In some embodiments, the cyclodextrin derivative in of theγPAMN/cyclodextrin complex and/or cyclodextrin/therapeutic agent complexis a compound of Formula III:

wherein R equals:

(a) (H)_(21-x) or (—(CH₂)₄—SO₃Na)x, and x=1.0-10.0, 1.0-5.0, 6.0-7.0, or8.0-10.0;

(b) (H)_(21-x) or (—(CH₂CH(OH)CH₃)x, and x=1.0-10.0, 1.0-5.0, 6.0-7.0,or 8.0-10.0;

(c) (H)_(21-x) or (sulfoalkyl ethers)x, and x=1.0-10.0, 1.0-5.0,6.0-7.0, or 8.0-10.0; or

(d) (H)_(21-x) or (—(CH₂)₄—SO₃Na)x, and x=1.0-10.0, 1.0-5.0, 6.0-7.0, or8.0-10.0.

In additional embodiments, the γPAMN/cyclodextrin complex and/orcyclodextrin/therapeutic agent complex is encapsulated in a liposome(e.g., as described herein or otherwise known in the art).

III. γPAMN Delivery Vehicles

In alternative embodiments, the disclosure provides γPAMN deliverysystems and their use to deliver a payload of γPAMN to a cell or cellsin vitro or in vivo. In some embodiments, γPAMN is complexed with orincorporated into a delivery vehicle. Such delivery vehicles are knownin the art and include, but are not limited to, liposomes, lipospheres,polymers, peptides, proteins, antibodies (e.g., ADCs such asAntibody-γPAMN conjugates), cellular components, cyclic oligosaccharides(e.g., cyclodextrins), nanoparticles (e.g., lipid nanoparticles,biodegradable nanoparticles, and core-shell nanoparticles), lipoproteinparticles, and combinations thereof. In particular embodiments, thedelivery vehicle is a liposome. In other particular embodiments, thedelivery vehicle is an antibody or an antigen binding antibody fragment.

A. Liposomes

In some embodiments, the disclosure provides liposomal compositions thatcomprise a liposome encapsulating (i.e., filled with) a gammapolyglutamated aminopterin (e.g., an γPAMN disclosed herein). In someembodiments, a liposome in the liposomal composition comprises a γPAMNcontaining 4, 5, 2-10, 4-6, or more than 5, glutamyl groups (includingthe glutamyl group in aminopterin). In some embodiments, the gammapolyglutamated aminopterin in the Lp-γPAMN comprises two or moreglutamyl groups in the L-form. In other embodiments, the gammapolyglutamated aminopterin in the Lp-γPAMN comprises a glutamyl group inthe D-form. In further embodiments, the gamma polyglutamated aminopterinin the Lp-γPAMN comprises a glutamyl group in the D-form and two or moreglutamyl groups in the L-form. In additional embodiments, the gammapolyglutamated aminopterin in the Lp-γPAMN comprises two or moreglutamyl groups that have a glamma carboxyl linkage. In someembodiments, the liposomal composition comprises a liposome comprising aγpentaglutamated AMN. In further embodiments, the liposome comprises anL-γpentaglutamated AMN, a D-γpentaglutamated AMN, or an L- andD-γpentaglutamated AMN. In some embodiments, the liposomal compositioncomprises a liposome comprising a γhexaglutamated AMN (Lp-γPAMN). Infurther embodiments, the liposome comprises an L-γhexaglutamated AMN, aD-γhexaglutamated AMN, or an L- and D-γhexaglutamated AMN. In someembodiments, the liposomal composition comprises a liposome that isanionic or neutral. In some embodiments, the liposomal compositioncomprises a liposome that is cationic. In some embodiments, the Lp-γPAMNcomposition is unpegylated. In some embodiments, the Lp-γPAMNcomposition is non-targeted (NTLp-γPAMN). In other embodiments, theLp-γPAMN composition is targeted (TLp-γPAMN). In some embodiments, theliposomal composition comprises a liposome having a diameter in therange of 20 nm to 500 nm, or any range therein between. In someembodiments, the liposomal composition comprises a liposome having adiameter in the range of 20 nm to 400 nm, or any range therein between.In some embodiments, the liposomal composition comprises a liposomehaving a diameter in the range of 20 nm to 300 nm or any range thereinbetween. In some embodiments, the liposomal composition comprises aliposome having a diameter in the range of 20 nm to 200 nm, or any rangetherein between. In further embodiments, the liposomal compositioncomprises a liposome having a diameter in the range of 20 nm to 150 nm,or any range therein between. In further embodiments, the liposomalcomposition comprises a liposome having a diameter in the range of 80 nmto 120 nm, or any range therein between. In additional embodiments,30-70%, 30-60%, or 30-50% w/w gamma polyglutamated aminopterin, or anyrange therein between, is encapsulated (entrapped) in the Lp-γPAMNduring the process of preparing the liposomes. In some embodiments, theLp-αPAMN composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%,35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of thegamma polyglutamated AMN. In some embodiments, at least 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or morethan 75%, w/w, gamma polyglutamated aminopterin, is encapsulated in theLp-γPAMN during the process of preparing the liposomes.

In some embodiments, the provided liposomes further comprise animmunostimulatory agent, a detectable marker, or both disposed on itsexterior. The immunostimulatory agent or detectable marker can beionically bonded or covalently bonded to an exterior of the liposome,including, for example, optionally to a steric stabilizer component ofthe liposome.

The terms “immunostimulatory agents”, also known as “immunostimulants”,and “immunostimulators”, refer to substances that stimulate an immune(including a preexisting immune response) by inducing activation orincreasing activity of any of the components of the immune system. Theseimmunostimulatory agents can include one or more of a hapten, anadjuvant, a protein immunostimulating agent, a nucleic acidimmunostimulating agent, and a chemical immunostimulating agent. Manyadjuvants contain a substance designed to stimulate immune responses,such as lipid A, Bortadella pertussis or Mycobacterium tuberculosisderived proteins. Certain adjuvants are commercially available as, forexample, Freund's Incomplete Adjuvant and Complete Adjuvant (DifcoLaboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company,Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.);aluminum salts such as aluminum hydroxide gel (alum) or aluminumphosphate; salts of calcium, iron or zinc; an insoluble suspension ofacylated tyrosine; acylated sugars; cationically or anionicallyderivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A; IFN-alpha, IFN-gamma,FLT3-ligand; and immunostimulatory antibodies (e.g., anti-CTLA-4,anti-CD28, anti-CD3. Cytokines, such as GM-CSF, interleukin-2, -7, -12,and -15, and other like growth factors, can also be used as adjuvants.In a preferred embodiment, the immunostimulant can be at least oneselected from the group consisting of fluorescein, DNP, beta glucan,beta-1,3-glucan, beta-1,6-glucan. In an additional preferred embodiment,the immunostimulant is a Toll-like receptor (TLR) modulating agent. Infurther embodiments, the Toll-like receptor (TLR) modulating agent isone or more of: an oxidized low-density lipoprotein (e.g., OXPAC, PGPC),an eritoran lipid (e.g., E5564), and a resolvin. In some embodiments,the liposomes comprise fluorescein isothiocyanate (FITC) which, based onour experiments, surprisingly serves as both an immunostimulant and adetectable marker.

In some embodiments, the liposomes comprise a detectable marker. Adetectable marker may, for example, include, at least, a radioisotope, afluorescent compound, a bioluminescent compound, chemiluminescentcompound, a metal chelator, an enzyme, a dye, an ink, a magneticcompound, a biocatalyst or a pigment that is detectable by any suitablemeans known in the art, e.g., magnetic resonance imaging (MRI), opticalimaging, fluorescent/luminescent imaging, and/or nuclear imagingtechniques.

In some embodiments, the immunostimulatory agent and/or detectablemarker is attached to the exterior by co-incubating it with theliposome. For example, the immunostimulatory agent and/or detectablemarker may be associated with the liposomal membrane by hydrophobicinteractions or by an ionic bond such as an avidin/biotin bond or ametal chelation bond (e.g., Ni-NTA). Alternatively, theimmunostimulatory agent or detectable marker may be covalently bonded tothe exterior of the liposome such as, for example, by being covalentlybonded to a liposomal component or to the steric stabilizer which is thePEG.

In some embodiments, the liposomes further comprise an agent thatincreases the uptake of liposomes into a cellular compartment ofinterest including the cytosol.

In some embodiments, the liposomes comprise a mitochondrial-targetingagent. In some embodiments, the liposomes comprise triphenylphosphonium(TPP). Methods and mechanisms for surface functionalizing liposomes withTPP are known in the art (e.g., attaching TPP to the lipid anchor via apeg spacer group and modifying TPP with a stearyl group (stearyltriphenylphosphonium (STPP)). In some embodiments, the liposomescomprise high-density octa-arginine. In some embodiments, the liposomescomprise sphingomyelin and/or a sphingomyelin metabolite. Sphingomyelinmetabolite used to formulate the liposomes of the present invention caninclude, for example ceramide, sphingosine or sphingosine 1-phosphate.In some embodiments, the liposomes comprise Rhodamine 123. In someembodiments, the liposomes comprise, a mitochondria penetrating peptide.In some embodiments, the liposomes comprise, a mitochondria penetratingagent selected from the group consisting of: a mitofusin peptide, amitochondrial targeting signal peptide, and Antennapedia helix IIIhomeodomain cell-penetrating peptide (ANT) (e.g., comprisingRQIKIWFQNRRMKWKKRKKRRQR RR (SEQ ID NO:1), RKKRRXR RRGC where X is anynatural or non-natural amino acid (SEQ ID NO:2), CCGCCAAGAAGCG (SEQ IDNO:3), GCGTGCACACGCGCGTAGACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTGAGCGGCGTGGCGCGGGGGCGTCAT (SEQ ID NO:4), ACGTGCATACGCACGTAGACATTCCCCGCTTCCCACTCCAAAGTCCGCCAAGAAGCGTATCCCGCTGAGCGGCGTGGCGCGGGGGCGTCATCCGTCAGCTC (SEQ ID NO:5), or ACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTG (SEQ ID NO:6)), or amitochondrial penetrating fragment thereof.

In some embodiments, liposomes in the provided liposome compositionscomprise a mitochondria penetrating agent selected from the group: aguanidine-rich peptoid, tetraguanidinium, triguanidinium, diguanidinium,monoguanidinium, a guanidine-rich polycarbamate, a beta-oligoarginine, aproline-rich dendrimer, and a phosphonium salt (e.g.,methyltriphenyl-phosphonium and/or tetraphenylphosphonium).

In some embodiments, liposomes in the provided liposome compositionscomprise sphingomyelin and/or stearyl-octa-arginine. In someembodiments, the liposomes comprise sphingomyelin and/orstearyl-octa-arginine. In some embodiments, the liposomes comprise DOPE,sphingomyelin, stearyl-octa-arginine sphingomyelin andstearyl-octa-arginine. In some embodiments, the liposomes comprise DOPE,sphingomyelin, stearyl-octa-arginine sphingomyelin andstearyl-octa-arginine at a molar ratio of 9:2:1. In some embodiments,the liposomes comprise the MITO-porter® system or a variant thereof.

In some embodiments, liposomes in the provided liposome compositionscomprise an agent such as a cell penetrating agent that that facilitatesdelivery of the liposome across a cell membrane and provides theliposome with the ability to bypass the endocytic pathway and the harshenvironment of lysosomes. Cell penetrating agents are known in the artand can routinely be used and adapted for manufacture and use of theprovided liposome compositions. In some embodiments, the cellpenetrating/lysosome bypassing agent is chloroquine. In someembodiments, the cell penetrating agent is a cell penetrating peptide.In some embodiments, liposomes in the provided liposome compositionscomprise a cell penetrating agent selected from the group: RKKRRQRRR(SEQ ID NO:7), GRKKRRQRRRTPQ (SEQ ID NO:8), YGRKKRRQRRR (SEQ ID NO:9),AAVAL LPAVLLALLA (SEQ ID NO:10), MGLGLHLLVLAAALQ (SEQ ID NO:11), GALFLGFLGAAGSTM (SEQ ID NO:12), AGYLLGKINLKALAALAKKIL (SEQ ID NO:13),RVIRVWFQNKRCKDKK (SEQ ID NO:14), RQIKIWFQNRRMKWKK (SEQ ID NO:15),GLFEAIAGFIENGWEGMIDG (SEQ ID NO:16), GWTLNSAGYLLGKIN (SEQ ID NO:17),RSQSRSRYYRQRQRS (SEQ ID NO:18), LAIPEQEY (SEQ ID NO:19), LGIAEQEY (SEQID NO:20), LGIPAQEY (SEQ ID NO:21), LGIPEAEY (SEQ ID NO:22), LGIPEQAY(SEQ ID NO:23), LGIAEAEY (SEQ ID NO:24), LGIPEAAY (SEQ ID NO:25),LGIAEQAY (SEQ ID NO:26), LGIAEAAY (SEQ ID NO:27), LLIILRRRIRKQAHAHSK(SEQ ID NO:28), LKALAALAKKIL (SEQ ID NO:29), KLALKLALKALKAALKLA (SEQ IDNO:30), KETWWETWWTEWSQPKKKRKV (SEQ ID NO:31), DHQLNPAF (SEQ ID NO:32),DPKGDPKG (SEQ ID NO:33), VTVTVTVTVTGKGDPKPD (SEQ ID NO:34),RQIKIWFQNRRMKWKK (SEQ ID NO:35), GRKKRRQRRRPPQ (SEQ ID NO:36),GWTLNSAGYLLGKINLKALAAL AKKIL (SEQ ID NO:37), GRKKRRQRRR (SEQ ID NO:38),RRRRRRR (SEQ ID NO:39), RRRRRRRR (SEQ ID NO:40), RRRRRRRRR (SEQ IDNO:41), RRRRRRRR RR (SEQ ID NO:42), RRRRRRRRRRR (SEQ ID NO:43), andYTIWMPENPRPGT PCDIFTNSRGKRASNGGG G(R)n wherein n=2-15 R in the L- and/orD-form (SEQ ID NO:44), or a cell permeating fragment thereof.

As discussed above, the liposomes may comprise a steric stabilizer thatcan increase their longevity in circulation. For those embodiments,which incorporate a steric stabilizer, the steric stabilizer may be atleast one member selected from the group consisting of polyethyleneglycol (PEG), poly-L-lysine (PLL), monosialoganglioside (GM1),poly(vinyl pyrrolidone) (PVP), poly(acrylamide) (PAA),poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), phosphatidylpolyglycerol, poly[N-(2-hydroxypropyl) methacrylamide], amphiphilicpoly-N-vinylpyrrolidones, L-amino-acid-based polymer, oligoglycerol,copolymer containing polyethylene glycol and polypropylene oxide,Poloxamer 188, and polyvinyl alcohol. In some embodiments, the stericstabilizer or the population of steric stabilizer is PEG. In oneembodiment, the steric stabilizer is a PEG. In a further embodiment, thePEG has a number average molecular weight (Mn) of 200 to 5000 daltons.These PEG(s) can be of any structure such as linear, branched, star orcomb structure and are commercially available.

In some embodiments, the liposomal composition comprises a pegylatedliposome (PLp-γPAMN). In some embodiments, a pegylated liposome in theliposomal composition comprises a γPAMN containing 4, 5, 2-10, 4-6, ormore than 5, glutamyl groups. In some embodiments, the gammapolyglutamated aminopterin in the Lp-γPAMN comprises two or moreglutamyl groups in the L-form. In other embodiments, the gammapolyglutamated aminopterin in the Lp-γPAMN comprises a glutamyl group inthe D-form. In further embodiments, the gamma polyglutamated aminopterinin the Lp-γPAMN comprises a glutamyl group in the D-form and two or moreglutamyl groups in the L-form. In some embodiments, the liposomalcomposition comprises a pegylated liposome comprising a γpentaglutamatedAMN. In further embodiments, the liposome comprises anL-γpentaglutamated AMN, a D-γpentaglutamated AMN, or an L- andD-γpentaglutamated AMN. In some embodiments, the liposomal compositioncomprises a pegylated liposome comprising a γhexaglutamated AMN. Infurther embodiments, the liposome comprises an L-γhexaglutamated AMN, aD-γhexaglutamated AMN, or an L- and D-γhexaglutamated AMN. In someembodiments, the liposomal composition comprises a pegylated liposomethat is anionic or neutral. In some embodiments, the liposomalcomposition comprises a pegylated liposome that is cationic. In someembodiments, the PLp-γPAMN composition is non-targeted (NTPLp-γPAMN). Inother embodiments, the PLp-γPAMN composition is targeted (TPLp-γPAMN).In some embodiments, the liposomal composition comprises at least 1%,5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,or more than 75%, w/w of the gamma polyglutamated aminopterin. In someembodiments, the liposomal composition comprises a pegylated liposomecomprising at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the gammapolyglutamated aminopterin is encapsulated (entrapped) in the PLp-γPAMNduring the process of preparing the liposomes. In some embodiments, theliposomal composition comprises a pegylated liposome having a diameterin the range of 20 nm to 500 nm. In some embodiments, the liposomalcomposition comprises a pegylated liposome having a diameter in therange of 20 nm to 400 nm. In some embodiments, the liposomal compositioncomprises a pegylated liposome having a diameter in the range of 20 nmto 300 nm. In some embodiments, the liposomal composition comprises apegylated liposome having a diameter in the range of 20 nm to 200 nm. Infurther embodiments, the liposomal composition comprises a pegylatedliposome having a diameter in the range of 80 nm to 120 nm.

In some embodiments, greater than 70%, 80% or 90% of the polyglutamatedaminopterin in a provided liposomal composition is pentaglutamated. Insome embodiments, greater than 70%, 80% or 90% of the polyglutamatedaminopterin in a provided composition is hexaglutamated. In someembodiments, greater than 70%, 80% or 90% of the polyglutamatedaminopterin in the composition has 4-10, 4-6, or more than 5, γ-glutamylgroups.

In some embodiments, the gamma polyglutamated aminopterin compositions(e.g., polyglutamates and delivery vehicles such as liposomes containingthe polyglutamates) are in an aqueous solution. In some embodiments, theγPAMN composition is administered in a liposomal composition at a doseof between 0.005 and 5000 mg of γPAMN per square meter (m²) of bodysurface area, or any range therein between. In further embodiments, theγPAMN composition is administered in a liposomal composition at a doseof between 0.1 and 1000 mg γPAMN per square meter of body surface area,or any range therein between.

(1) Liposome Composition

The lipids and other components of the liposomes contained in theliposomal compositions can be any lipid, lipid combination and ratio, orcombination of lipids and other liposome components and their respectiveratios known in the art. However, it will be understood by one skilledin the art that liposomal encapsulation of any particular drug, such as,and without limitation, the gamma polyglutamated AMN discussed herein,may involve substantial routine experimentation to achieve a useful andfunctional liposomal formulation. In general, the provided liposomes mayhave any liposome structure, e.g., structures having an inner spacesequestered from the outer medium by one or more lipid bilayers, or anymicrocapsule that has a semi-permeable membrane with a lipophiliccentral part where the membrane sequesters an interior. The lipidbilayer can be any arrangement of amphiphilic molecules characterized bya hydrophilic part (hydrophilic moiety) and a hydrophobic part(hydrophobic moiety). Usually amphiphilic molecules in a bilayer arearranged into two dimensional sheets in which hydrophobic moieties areoriented inward the sheet while hydrophilic moieties are orientedoutward. Amphiphilic molecules forming the provided liposomes can be anyknown or later discovered amphiphilic molecules, e.g., lipids ofsynthetic or natural origin or biocompatible lipids. The liposomes canalso be formed by amphiphilic polymers and surfactants, e.g.,polymerosomes and niosomes. For the purpose of this disclosure, withoutlimitation, these liposome-forming materials also are referred to as“lipids”.

The liposome composition formulations provided herein can be in liquidor dry form such as a dry powder or dry cake. The dry powder or dry cakemay have undergone primary drying under, for example, lyophilizationconditions or optionally, the dry cake or dry powder may have undergoneboth primary drying only or both primary drying and secondary drying. Inthe dry form, the powder or cake may, for example, have between 1% to 6%moisture, for example, such as between 2% to 5% moisture or between 2%to 4% moisture. One example method of drying is lyophilization (alsocalled freeze-drying, or cyrodessication). Any of the compositions andmethods of the disclosure may include liposomes, lyophilized liposomesor liposomes reconstituted from lyophilized liposomes. In someembodiments, the disclosed compositions and methods include one or morelyoprotectants or cryoprotectants. These protectants are typicallypolyhydroxy compounds such as sugars (mono-, di-, and polysaccharides),polyalcohols, and their derivatives, glycerol, or polyethyleneglycol,trehalose, maltose, sucrose, glucose, lactose, dextran, glycerol, oraminoglycosides. In further embodiments, the lyoprotectants orcryoprotectants comprise up to 10% or up to 20% of a solution outsidethe liposome, inside the liposome, or both outside and inside theliposome.

In some embodiments, the liposomes include a steric stabilizer thatincreases their longevity in circulation. One or more steric stabilizerssuch as a hydrophilic polymer (Polyethylene glycol (PEG)), a glycolipid(monosialoganglioside (GM1)) or others occupies the space immediatelyadjacent to the liposome surface and excludes other macromolecules fromthis space. Consequently, access and binding of blood plasma opsonins tothe liposome surface are hindered, and thus interactions of macrophageswith such liposomes, or any other clearing mechanism, are inhibited andlongevity of the liposome in circulation is enhanced. In someembodiments, the steric stabilizer or the population of stericstabilizers is a PEG or a combination comprising PEG. In furtherembodiments, the steric stabilizer is a PEG or a combination comprisingPEG with a number average molecular weight (Mn) of 200 to 5000 daltons.These PEG(s) can be of any structure such as linear, branched, star orcomb structure and are commercially available.

The diameter of the disclosed liposomes is not particularly limited. Insome embodiments, the liposomes have a diameter in the range of forexample, 30-150 nm (nanometer). In other embodiments, the liposomes havea diameter in the range of 40-70 nm.

The properties of liposomes are influenced by the nature of lipids usedto make the liposomes. A wide variety of lipids have been used to makeliposomes. These include cationic, anionic and neutral lipids. In someembodiments, the liposomes comprising the gamma polyglutamatedaminopterin are anionic or neutral. In other embodiments, the providedliposomes are cationic. The determination of the charge (e.g., anionic,neutral or cationic) can routinely be determined by measuring the zetapotential of the liposome. The zeta potential of the liposome can bepositive, zero or negative. In some embodiments, the zeta potential ofthe liposome is less than or equal to zero. In some embodiments, thezeta potential of the liposome is in a range of 0 to −150 mV. In anotherembodiment, the zeta potential of the liposome is in the range of −30 to−50 mV.

In some embodiments, cationic lipids are used to make cationic liposomeswhich are commonly used as gene transfection agents. The positive chargeon cationic liposomes enables interaction with the negative charge oncell surfaces. Following binding of the cationic liposomes to the cell,the liposome is transported inside the cell through endocytosis.

In some preferred embodiments, a neutral to anionic liposome is used. Ina preferred embodiment, an anionic liposome is used. Using a mixture of,for example, neutral lipids such as HSPC and anionic lipids such asPEG-DSPE results in the formation of anionic liposomes which are lesslikely to non-specifically bind to normal cells. Specific binding totumor cells can be achieved by using a tumor targeting antibody such as,for example, a folate receptor antibody, including, for example, folatereceptor alpha antibody, folate receptor beta antibody and/or folatereceptor delta antibody.

As an example, at least one (or some) of the lipids is/are amphipathiclipids, defined as having a hydrophilic and a hydrophobic portions(typically a hydrophilic head and a hydrophobic tail). The hydrophobicportion typically orients into a hydrophobic phase (e.g., within thebilayer), while the hydrophilic portion typically orients toward theaqueous phase (e.g., outside the bilayer). The hydrophilic portion cancomprise polar or charged groups such as carbohydrates, phosphate,carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxy and other likegroups. The hydrophobic portion can comprise apolar groups that includewithout limitation long chain saturated and unsaturated aliphatichydrocarbon groups and groups substituted by one or more aromatic,cyclo-aliphatic or heterocyclic group(s). Examples of amphipathiccompounds include, but are not limited to, phospholipids, aminolipidsand sphingolipids.

Typically, for example, the lipids are phospholipids. Phospholipidsinclude without limitation phosphatidylcholine,phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol,phosphatidylserine, and the like. It is to be understood that otherlipid membrane components, such as cholesterol, sphingomyelin, andcardiolipin, can be used.

The lipids comprising the liposomes provided herein can be anionic andneutral (including zwitterionic and polar) lipids including anionic andneutral phospholipids. Neutral lipids exist in an uncharged or neutralzwitterionic form at a selected pH. At physiological pH, such lipidsinclude, for example, dioleoylphosphatidylglycerol (DOPG),diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols.Examples of zwitterionic lipids include without limitationdioleoylphosphatidylcholine (DOPC), dimyristoylphos-phatidylcholine(DMPC), and dioleoylphosphatidylserine (DOPS). Anionic lipids arenegatively charged at physiological pH. These lipids include withoutlimitation phosphatidylglycerol, cardiolipin, diacylphosphatidylserine,diacylphosphatidic acid, N-dode-canoyl phosphatidylethanolamines,N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphos-phatidylglycerol(POPG), and other anionic modifying groups joined to neutral lipids.

Collectively, anionic and neutral lipids are referred to herein asnon-cationic lipids. Such lipids may contain phosphorus but they are notso limited. Examples of non-cationic lipids include lecithin,lysolecithin, phosphatidylethanolamine, lysophosphatidylethan-olamine,dioleoylphosphati-dylethanolamine (DOPE), dipalmitoyl phosphatidylethanol-amine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidy 1-ethan-olamine (DSPE),palmitoyloleoyl-phosphatidylethanolamine (POPE)palmitoyl-oleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), distearoylphosphat-idylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphospha-tidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphospha-tidylglycerol (DPPG),palmitoyloleyolphosphatidylglycerol (POPG), 16-0-monomethyl PE,16-0-dimethyl PE, 18-1-trans PE,palmitoyloleoyl-phosphatidylethanolamine (POPE),1-stearoyl-2-oleoylphosphatidyethanolamine (SOPE), phosphatidylserine,phosphatidyl-inositol, sphingomyelin, cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetyl-phosphate, and cholesterol.

The liposomes may be assembled using any liposomal assembly method usingliposomal components (also referred to as liposome components) known inthe art. Liposomal components include, for example, lipids such as DSPE,HSPC, cholesterol and derivatives of these components. Other suitablelipids are commercially available for example, by Avanti Polar Lipids,Inc. (Alabaster, Ala., USA). A partial listing of available negativelyor neutrally charged lipids suitable for making anionic liposomes, canbe, for example, at least one of the following: DLPC, DMPC, DPPC, DSPC,DOPC, DMPE, DPPE, DOPE, DMPA.Na, DPPA.Na, DOPA.Na, DMPG.Na, DPPG.Na,DOPG.Na, DMPS.Na, DPPS.Na, DOPS.Na, DOPE-Glutaryl.(Na)2, TetramyristoylCardiolipin.(Na)2, DSPE-mPEG-2000.Na, DSPE-mPEG-5000.Na, andDSPE-Maleimide PEG-2000.Na.

In some embodiments, the γPAMN compositions provided herein areformulated in a liposome comprising a cationic lipid. In one embodiment,the cationic lipid is selected from, but not limited to, a cationiclipid described in Intl. Appl. Publ. Nos. WO2012/040184, WO2011/153120,WO2011/149733, WO2011/090965, WO2011/043913, WO2011/022460,WO2012/061259, WO2012/054365, WO2012/044638, WO2010/080724, WO2010/21865and WO2008/103276, U.S. Pat. Nos. 7,893,302, 7,404,969 and 8,283,333 andUS Appl. Publ. Nos. US20100036115 and US20120202871; each of which isherein incorporated by reference in their entirety. In anotherembodiment, the cationic lipid may be selected from, but not limited to,formula A described in Intl. Appl. Publ. Nos. WO2012/040184,WO2011/153120, WO201/1149733, WO2011/090965, WO2011/043913,WO2011/022460, WO2012/061259, WO2012/054365 and WO2012/044638; each ofwhich is herein incorporated by reference in their entirety. In yetanother embodiment, the cationic lipid may be selected from, but notlimited to, formula CLI-CLXXIX of International Publication No.WO2008103276, formula CLI-CLXXIX of U.S. Pat. No. 7,893,302, formulaCLI-CLXXXXII of U.S. Pat. No. 7,404,969 and formula I-VI of US PatentPublication No. US20100036115; each of which is herein incorporated byreference in their entirety. As a non-limiting example, the cationiclipid may be selected from(20Z,23Z)—N,N-dimethylnonacosa-20,23-dien-10-amine,(17Z,20Z)—N,N-dimemyl-hexacosa-17,20-dien-9-amine,(1Z,19Z)—N5N-dimethylpentacosa-16, 19-dien-8-amine,(13Z,16Z)—N,N-dimethyldocosa-13,16-dien-5-amine,(12Z,15Z)—N,N-dimethylhenicosa-12,15-dien-4-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-6-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-7-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-10-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-5-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-4-amine,(19Z,22Z)—N,N-dimeihyloctacosa-19,22-dien-9-amine, (18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-8-amine,(17Z,20Z)—N,N-dimethylhexacosa-17,20-dien-7-amine,(16Z,19Z)—N,N-dimethylpentacosa-16,19-dien-6-amine,(22Z,25Z)—N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z,24Z)—N,N-dimethyl-triaconta-21,24-dien-9-amine,(18Z)—N,N-dimetylheptacos-18-en-10-amine,(17Z)—N,N-dimethylhexacos-17-en-9-amine,(19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-7-amine,N,N-dimethylheptacosan-10-amine,(20Z,23Z)—N-ethyl-N-methylnonacosa-20,23-dien-10-amine,1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl] pyrrolidine,(20Z)—N,N-dimethylheptacos-20-en-10-amine, (15Z)—N,N-dimethyleptacos-15-en-10-amine, (14Z)—N,N-dimethylnonacos-14-en-10-amine,(17Z)—N,N-dimethylnonacos-17-en-10-amine,(24Z)—N,N-dimethyltritriacont-24-en-10-amine,(20Z)—N,N-dimethylnonacos-20-en-10-amine,(22Z)—N,N-dimethylhentriacont-22-en-10-amine,(16Z)—N,N-dimethylpenta-cos-16-en-8-amine,(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl] eptadecan-8-amine,1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethyl nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine,N,N-dimethyl-21-[R₁S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine,N,N-dimethyl-3-17-[(1S,2R)-2-octylcyclopropyl]heptyl 1 dodecan-1-amine,1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethyl-penta-decan-6-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine,R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propa-n-2-amine,S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine,(2S)—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z-)-oct-5-en-1-yloxy]propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl] ethyl}azetidine,(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-ylo-xy]propan-2-amine,(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]pr-opan-2-amine,N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine;(2S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine,(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)pro-pan-2-amine,(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylprop-an-2-amine,1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl1-3-(octyloxy)propan-2-amine,1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dime-thylpropan-2-amine,(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy) propan-2-amine,(2R)—N,N-dimethyl-H(1-metoyloctyl)oxyl-3-[(9Z,12Z)-octa-deca-9,12-dien-1-yloxy]propan-2-amine,(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-R9Z,12Z)-octadeca-9,12-die-n-1-yloxylpropan-2-amine,N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]-methyl}cyclopropyl]octyl} oxy) propan-2-amine,N,N-dimethyl-1-{[-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and(11E,20Z,23Z)—N,N-dimethylnonacosa-11,20,2-trien-10-amine or apharmaceutically acceptable salt or acid or stereoisomer thereof.

In one embodiment, the lipid may be a cleavable lipid such as thosedescribed in in Intl. Publ. No. WO2012/170889, which is hereinincorporated by reference in its entirety

The cationic lipid can routinely be synthesized using methods known inthe art and/or as described in Intl. Publ. Nos. WO2012/040184,WO2011/153120, WO2011/149733, WO2011/090965, WO201/1043913,WO2011/022460, WO2012/061259, WO2012/054365, WO2012/044638,WO2010/080724 and WO2010/21865; each of which is herein incorporated byreference in its entirety.

Lipid derivatives can include, for example, at least, the bonding(preferably covalent bonding) of one or more steric stabilizers and/orfunctional groups to the liposomal component after which the stericstabilizers and/or functional groups should be considered part of theliposomal components. Functional groups comprises groups that can beused to attach a liposomal component to another moiety such as aprotein. Such functional groups include, at least, maleimide. Thesesteric stabilizers include at least one from the group consisting of:polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside(GM1); poly(vinyl pyrrolidone) (PVP); poly(acrylamide) (PAA);poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidylpolyglycerol; poly[N-(2-hydroxy-propyl) methacrylamide]; amphiphilicpoly-N-vinylpyrrolidones; L-amino-acid-based polymer; and polyvinylalcohol.

In some embodiments, the γPAMN compositions are formulated in alipid-polycation complex. The formation of the lipid-polycation complexmay be accomplished using methods known in the art and/or as describedin U.S. Pub. No. 20120178702, herein incorporated by reference in itsentirety. As a non-limiting example, the polycation may include acationic peptide or a polypeptide such as, but not limited to,polylysine, polyomithine and/or polyarginine and the cationic peptidesdescribed in International Pub. No. WO2012/013326; herein incorporatedby reference in its entirety. In another embodiment, the γPAMN isformulated in a lipid-polycation complex which further includes aneutral lipid such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

Since the components of a liposome can include any molecule(s) (e.g.,chemical/reagent/protein) that is bound to it, in some embodiments, thecomponents of the provided liposomes include, at least, a memberselected from the group DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG;HSPC-maleimide; cholesterol; cholesterol-PEG; and cholesterol-maleimide.In some embodiments, the components of the provided liposomes includeDSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC-maleimide;cholesterol; cholesterol-PEG; and cholesterol-maleimide. In a preferredembodiment, the liposomal components that make up the liposome comprisesDSPE; DSPE-FITC; DSPE-maleimide; cholesterol; and HSPC.

In additional embodiments, the liposomes of the liposome compositionsprovided herein comprise oxidized phospholipids. In some embodiments,the liposomes comprise an oxidize phospholipid of a member selected fromthe group consisting of phosphatidylserines, phosphatidylinositols,phosphatidylethanolamines, phosphatidyl-cholines and1-palmytoyl-2-arachidonoyl-sn-glycero-2-phosphate. In some embodiments,the phospholipids have unsaturated bonds. In some embodiments, thephospholipids are arachidonic acid containing phospholipids. Inadditional embodiments, the phospholipids are sn-2-oxygenated. Inadditional embodiments, the phospholipids are not fragmented.

In some embodiments, the liposomes of the disclosed liposomecompositions comprise oxidized1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC). Theterm “oxPAPC”, as used herein, refers to lipids generated by theoxidation of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine(PAPC), which results in a mixture of oxidized phospholipids containingeither fragmented or full length oxygenated sn-2 residues.Well-characterized oxidatively fragmented species contain a five-carbonsn-2 residue bearing omega-aldehyde or omega-carboxyl groups. Oxidationof arachidonic acid residue also produces phospholipids containingesterified isoprostanes. OxPAPC includes HOdiA-PC, KOdiA-PC, HOOA-PC andKOOA-PC species, among other oxidized products present in oxPAPC. Infurther embodiments, the oxPAPCs are epoxyisoprostane-containingphospholipids. In further embodiments, the oxPAPC is1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine(5,6-PEIPC),1-palmitoyl-2-(epoxy-cyclo-pentenone)-sn-glycero-3-phosphorylcholine(PECPC) and/or 1-palmitoyl-2-(epoxy-isoprostaneE2)-sn-glycero-4-phosphocholine (PEIPC). In some embodiments, thephospholipids have unsaturated bonds. In some embodiments, thephospholipids are arachidonic acid containing phospholipids. Inadditional embodiments, the phospholipids are sn-2-oxygenated. Inadditional embodiments, the phospholipids are not fragmented.

In some embodiments, the liposomal gamma polyglutamated aminopterincomposition is pegylated (i.e., a pegylated liposomal gammapolyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate(PLp-γPAMN or TPLp-γPAMN)). In some embodiments, the PLp-γPAMN orTPLp-γPAMN is water soluble. That is, the PLp-γPAMN or TPLp-γPAMN is inthe form an aqueous solution.

In some embodiments, the liposomes of the disclosed liposomecompositions comprise a lipid selected from:1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC);1-palmitoyl-2-(9′oxo-nonanoyl)-sn-glycero-3-phosphocholine;1-palmitoyl-2-arachinodoyl-sn-glycero-3-phosphocholine;1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine;1-palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine;1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine; and1-palmitoyl-2-acetoyl-sn-glycero-3-phospho-choline. In furtherembodiments, the liposome comprises PGPC.

In some embodiments, the pH of solutions comprising the liposomecomposition is from pH 5 to 8 or from pH 2 to 6.2 to 8, or any rangetherein between. In some embodiments, the pH of solutions comprising theliposome composition is from pH 5 to 8, or any range therein between. Insome embodiments, the pH of solutions comprising the liposomecomposition is from pH 6 to 7, or any range therein between. In someembodiments, the pH of solutions comprising the liposome composition isfrom 6 to 7.5, from 6.5 to 7.5, from 6.7 to 7.5, or from 6.3 to 7.0, orany range therein between.

In some embodiments, at least one component of the liposome lipidbilayer is functionalized (or reactive). As used herein, afunctionalized component is a component that comprises a reactive groupthat can be used to crosslink reagents and moieties to the lipid. If thelipid is functionalized, any liposome that it forms is alsofunctionalized. In some embodiments, the reactive group is one that willreact with a crosslinker (or other moiety) to form crosslinks. Thereactive group in the liposome lipid bilayer is located anywhere on thelipid that allows it to contact a crosslinker and be crosslinked toanother moiety (e.g., a steric stabilizer or targeting moiety). In someembodiments, the reactive group is in the head group of the lipid,including for example a phospholipid. In some embodiments, the reactivegroup is a maleimide group. Maleimide groups can be crosslinked to eachother in the presence of dithiol crosslinkers including but not limitedto dithiolthrietol (DTT).

It is to be understood that the use of other functionalized lipids,other reactive groups, and other crosslinkers beyond those describedabove is further contemplated. In addition to the maleimide groups,other examples of contemplated reactive groups include but are notlimited to other thiol reactive groups, amino groups such as primary andsecondary amines, carboxyl groups, hydroxyl groups, aldehyde groups,alkyne groups, azide groups, carbonyls, halo acetyl (e.g., iodoacetyl)groups, imidoester groups, N-hydroxysuccinimide esters, sulfhydrylgroups, and pyridyl disulfide groups.

Functionalized and non-functionalized lipids are available from a numberof commercial sources including Avanti Polar Lipids (Alabaster, Ala.)and Lipoid LLC (Newark, N.J.).

(2) Liposome Interior Space

In further non-limiting embodiments, the provided liposomes enclose aninterior space. In some embodiments, the interior space comprises, butis not limited to, an aqueous solution. In some embodiments, theinterior space comprises a gamma polyglutamated aminopterin as providedherein. In additional embodiments, the interior space of the liposomecomprises a tonicity agent. In some embodiments. In some embodiments,the concentration (weight percent) of the tonicity agent is 0.1-20%,1-20%, 0.5-15%, 1-15%, or 1-50%, or any range therein between. In someembodiments, the interior space of the liposome includes a sugar (e.g.,trehalose, maltose, sucrose, lactose, mannose, mannitol, glycerol,dextrose, fructose, etc.). In further embodiments, the concentration(weight percent) of the sugar is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or1-50%, or any range therein between. In some embodiments, the pH of theinterior space of the liposome is from pH 2 to 8, or any range thereinbetween. In some embodiments, the pH of solutions comprising theliposome composition is from pH 5 to 8, or any range therein between. Insome embodiments, the pH of solutions comprising the liposomecomposition is from pH 6 to 7, or any range therein between. In someembodiments, the pH of solutions comprising the liposome composition isfrom 6 to 7.5, from 6.5 to 7.5, from 6.7 to 7.5, or from 6.3 to 7.0, orany range therein between. In some embodiments, the interior spacecomprises buffer. In further embodiments, the buffer a buffer selectedfrom HEPES, citrate, or sodium phosphate (e.g., monobasic and/or dibasicsodium phosphate). In some embodiments, the buffer is HEPES. In someembodiments, the buffer is citrate. In some embodiments, the buffer issodium phosphate (e.g., monobasic and/or dibasic sodium phosphate). Insome embodiments, the buffer is at a concentration of 15 to 200 mM, orany range therein between. In yet further embodiments, the buffer is ata concentration of between 5 to 200 mM, 15-200, between 5 to 100 mM,between 15 to 100 mM, between 5 to 50 mM, between 15 to 50 mM, between 5to 25 mM, between 5 to 20 mM, between 5 to 15 mM, or any range thereinbetween. In some embodiments, the buffer is HEPES at a concentration of15 to 200 mM, or any range therein between. In some embodiments, thebuffer is citrate at a concentration of 15 to 200 mM, or any rangetherein between. In some embodiments, the buffer is sodium phosphate ata concentration of 15 to 200 mM, or any range therein between. In someembodiments, the interior space of the liposome comprises a totalconcentration of sodium acetate and calcium acetate of between 5 mM to500 mM, or 50 mM to 500 mM, or any range therein between.

In some embodiments, the interior space of the liposome includestrehalose. In further embodiments, the concentration weight percent oftrehalose is 0.1-20%, 1-20%, 0.5-15%, 1%-15%, or 5-20%, or any rangetherein between. In yet further embodiments, the concentration (weightpercent) of trehalose is 1-15%, or any range therein between. In anadditional embodiment, the trehalose is present at about 5% to 20%weight percent of trehalose or any combination of one or morelyoprotectants or cryoprotectants at a total concentration of 5% to 20%.In some embodiments, the pH of solutions comprising the liposomecomposition is from 6 to 7.5, from 6.5 to 7.5, from 6.7 to 7.5, or from6.3 to 7.0, or any range therein between. In some embodiments, theinterior space comprises buffer. In some embodiments, the buffer isselected from HEPES, citrate, or sodium phosphate (e.g., monobasicand/or dibasic sodium phosphate). In some embodiments, the buffer isHEPES. In some embodiments, the buffer is citrate. In some embodiments,the buffer is sodium phosphate (e.g., monobasic and/or dibasic sodiumphosphate). In some embodiments, the buffer is at a concentration of 15to 200 mM, or any range therein between. In yet further embodiments, thebuffer is at a concentration of between 5 to 200 mM, 15-200, between 5to 100 mM, between 15 to 100 mM, between 5 to 50 mM, between 15 to 50mM, between 5 to 25 mM, between 5 to 20 mM, between 5 to 15 mM, or anyrange therein between. In some embodiments, the buffer is HEPES at aconcentration of 15 to 200 mM, or any range therein between. In someembodiments, the buffer is citrate at a concentration of 15 to 200 mM,or any range therein between. In some embodiments, the buffer is sodiumphosphate at a concentration of 15 to 200 mM, or any range thereinbetween. In additional embodiments, the interior space of the liposomecomprises sodium acetate and/or calcium acetate. In some embodiments,the interior space of the liposome comprises a total concentration ofsodium acetate and calcium acetate of between 5 mM to 500 mM, or 50 mMto 500 mM, or any range therein between.

In some embodiments, the interior space of the liposome includesdextrose. In further embodiments, the concentration weight percent ofdextrose is 0.1-20%, 1-20%, 0.5-15%, 1%-15%, or 5-20%, or any rangetherein between. In yet further embodiments, the concentration (weightpercent) of dextrose is 1-15%, or any range therein between. In anadditional embodiment, the dextrose is present at about 5% to 20% weightpercent of dextrose or any combination of one or more lyoprotectants orcryoprotectants at a total concentration of 5% to 20%. In someembodiments, the pH of solutions comprising the liposome composition isfrom 6 to 7.5, from 6.5 to 7.5, from 6.7 to 7.5, or from 6.3 to 7.0, orany range therein between. In some embodiments, the interior spacecomprises buffer. In some embodiments, the buffer is selected fromHEPES, citrate, or sodium phosphate (e.g., monobasic and/or dibasicsodium phosphate). In some embodiments, the buffer is HEPES. In someembodiments, the buffer is citrate. In some embodiments, the buffer issodium phosphate (e.g., monobasic and/or dibasic sodium phosphate). Insome embodiments, the buffer is at a concentration of 15 to 200 mM, orany range therein between. In yet further embodiments, the buffer is ata concentration of between 5 to 200 mM, 15-200, between 5 to 100 mM,between 15 to 100 mM, between 5 to 50 mM, between 15 to 50 mM, between 5to 25 mM, between 5 to 20 mM, between 5 to 15 mM, or any range thereinbetween. In some embodiments, the buffer is HEPES at a concentration of15 to 200 mM, or any range therein between. In some embodiments, thebuffer is citrate at a concentration of 15 to 200 mM, or any rangetherein between. In some embodiments, the buffer is sodium phosphate ata concentration of 15 to 200 mM, or any range therein between Inadditional embodiments, the interior space of the liposome comprisessodium acetate and/or calcium acetate. In some embodiments, the interiorspace of the liposome comprises a total concentration of sodium acetateand calcium acetate of between 5 mM to 500 mM, or 50 mM to 500 mM, orany range therein between.

In additional embodiments, the disclosure provides liposomalcompositions that comprise a liposome encapsulating (filled with) agamma polyglutamated aminopterin (e.g., a γPAMN disclosed herein). Insome embodiments, a liposome in the liposomal composition comprises aγPAMN containing 4, 5, 2-10, 4-6, or more than 5, γ-glutamyl groups(including the glutamyl group in aminopterin). In some embodiments, thegamma polyglutamated aminopterin in the Lp-γPAMN comprises two or moreglutamyl groups in the L-form. In other embodiments, the gammapolyglutamated aminopterin in the Lp-γPAMN comprises a glutamyl group inthe D-form. In further embodiments, the gamma polyglutamated aminopterinin the Lp-γPAMN comprises a glutamyl group in the D-form and two or moreglutamyl groups in the L-form. In additional embodiments, the gammapolyglutamated aminopterin in the Lp-γPAMN comprises two or moreglutamyl groups that have a glamma carboxyl linkage. In someembodiments, the liposomal composition comprises a liposome comprising aγpentaglutamated AMN. In further embodiments, the liposome comprises anL-γpentaglutamated AMN, a D-γpentaglutamated AMN, or an L- andD-γpentaglutamated AMN. In some embodiments, the liposomal compositioncomprises a liposome comprising a γhexaglutamated AMN (Lp-γPAMN). Infurther embodiments, the liposome comprises an L-γhexaglutamated AMN, aD-γhexaglutamated AMN, or an L- and D-γhexaglutamated AMN.

In some embodiments, the targeted pegylated liposomal gammapolyglutamated (e.g., pentaglutamated or hexaglutamated) aminopterincomprises a medium comprising a liposome including an interior space; anaqueous gamma polyglutamated aminopterin disposed within the interiorspace; and a targeting moiety comprising a protein with specificaffinity for at least one folate receptor, and wherein the targetingmoiety disposed at the exterior of the liposome. In some embodiments,the medium is an aqueous solution. In some embodiments, the interiorspace, the exterior space (e.g., the medium), or both the interior spaceand the medium contains one or more lyoprotectants or cryoprotectantswhich are listed above. In some embodiments, the cryoprotectant ismannitol, trehalose, sorbitol, or sucrose.

In some embodiments, the liposome encapsulating gamma polyglutamatedaminopterin (i.e., Lp-γPAMN, including PLp-γPAMN, TPLp-γPAMN, TLp-γPAMN,and NTLp-γPAMN) has an interior space that contains less than 500,000 orless than 200,000 molecules of gamma polyglutamated aminopterin. In someembodiments, the liposome interior space contains between 10 to 100,000molecules of gamma polyglutamated aminopterin, or any range thereinbetween. In some embodiments, the liposome interior space containsbetween 10,000 to 100,000 molecules of gamma polyglutamated aminopterin,or any range therein between. In some embodiments, the liposome isunpegylated and has an interior space that contains less than 500,000 orless than 200,000 molecules of gamma polyglutamated aminopterin. In someembodiments, the liposome is unpegylated and the interior space of theliposome contains between 10 to 100,000 molecules of gammapolyglutamated aminopterin, or any range therein between. In furtherembodiments, the liposome is unpegylated and the interior space of theliposome contains between 10,000 to 100,000 molecules of gammapolyglutamated aminopterin, or any range therein between. In someembodiments, the liposome is targeted and unpegylated (TLp-γPAMN) andhas an interior space that contains less than 500,000 or less than200,000 molecules of gamma polyglutamated aminopterin. In someembodiments, the liposome is targeted and unpegylated and the interiorspace of the liposome contains between 10 to 100,000 molecules of gammapolyglutamated aminopterin, or any range therein between. In furtherembodiments, the liposome is targeted and unpegylated and the interiorspace of the liposome contains between 10,000 to 100,000 molecules ofgamma polyglutamated aminopterin, or any range therein between. In someembodiments, the liposome is non-targeted and unpegylated (NTLp-γPAMN)and has an interior space that contains less than 500,000 or less than200,000 molecules of gamma polyglutamated aminopterin. In someembodiments, the liposome is non-targeted and unpegylated and theinterior space of the liposome contains between 10 to 100,000 moleculesof gamma polyglutamated aminopterin, or any range therein between. Infurther embodiments, the liposome is non-targeted and unpegylated andthe interior space of the liposome contains between 10,000 to 100,000molecules of gamma polyglutamated aminopterin, or any range thereinbetween.

In some embodiments, the liposome encapsulates gamma polyglutamatedcontaining 2-10 glutamyl groups (i.e., Lp-γPAMN, including PLp-γPAMN,TPLp-γPAMN, TLp-γPAMN, and NTLp-γPAMN) and has an interior space thatcontains less than 500,000 or 200,000 molecules of gamma polyglutamatedaminopterin containing 2-10 glutamyl groups. In some embodiments, theliposome interior space contains between 10 to 100,000 molecules ofgamma polyglutamated aminopterin containing 2-10 glutamyl groups, or anyrange therein between. In further embodiments, the liposome interiorspace contains between 10,000 to 100,000 molecules of gammapolyglutamated aminopterin containing 2-10 glutamyl groups, or any rangetherein between. In some embodiments, the liposome is unpegylated andhas an interior space that contains less than 500,000 or 200,000molecules of gamma polyglutamated aminopterin containing 2-10 glutamylgroups. In some embodiments, the liposome is unpegylated and theinterior space of the liposome contains between 10 to 100,000 moleculesof gamma polyglutamated aminopterin containing 2-10 glutamyl groups, orany range therein between. In further embodiments, the liposome isunpegylated and the interior space of the liposome contains between10,000 to 100,000 molecules of gamma polyglutamated aminopterincontaining 2-10 glutamyl groups, or any range therein between. In someembodiments, the liposome is targeted and unpegylated (TLp-γPAMN) andhas an interior space that contains less than 500,000 or 200,000molecules of gamma polyglutamated aminopterin containing 2-10 glutamylgroups. In some embodiments, the liposome is targeted and unpegylatedand the interior space of the liposome contains between 10 to 100,000molecules gamma polyglutamated aminopterin containing 2-10 glutamylgroups, or any range therein between. In further embodiments, theliposome is targeted and unpegylated and the interior space of theliposome contains between 10,000 to 100,000 molecules gammapolyglutamated aminopterin containing 2-10 glutamyl groups, or any rangetherein between. In some embodiments, the liposome is non-targeted andunpegylated (NTLp-γPAMN) and has an interior space that contains lessthan 500,000 or 200,000 molecules of gamma polyglutamated aminopterincontaining 2-10 glutamyl groups. In some embodiments, the liposome isnon-targeted and unpegylated and the interior space of the liposomecontains between 10 to 100,000 molecules of gamma polyglutamatedaminopterin containing 2-10 glutamyl groups, or any range thereinbetween. In further embodiments, the liposome is non-targeted andunpegylated and the interior space of the liposome contains between10,000 to 100,000 molecules of gamma polyglutamated aminopterincontaining 2-10 glutamyl groups, or any range therein between.

In some embodiments, the liposome encapsulates gamma tetraglutamatedaminopterin (i.e., Lp-γPAMN, including PLp-γPAMN, TPLp-γPAMN, TLp-γPAMN,and NTLp-γPAMN) and has an interior space that contains less than500,000 or 200,000 molecules of gamma tetraglutamated aminopterin. Insome embodiments, the liposome interior space contains between 10 to100,000 molecules of gamma tetraglutamated aminopterin, or any rangetherein between. In some embodiments, the liposome interior spacecontains between 10,000 to 100,000 molecules of gamma tetraglutamatedaminopterin, or any range therein between. In some embodiments, theliposome is unpegylated and has an interior space that contains lessthan 500,000 or 200,000 molecules of gamma tetraglutamated aminopterin.In some embodiments, the liposome is unpegylated and the interior spaceof the liposome contains between 10 to 100,000 molecules of gammatetraglutamated aminopterin, or any range therein between. In furtherembodiments, the liposome is unpegylated and the interior space of theliposome contains between 10,000 to 100,000 molecules of gammatetraglutamated aminopterin, or any range therein between. In someembodiments, the liposome is targeted and unpegylated (TLp-γPAMN) andhas an interior space that contains less than 500,000 or 200,000molecules of gamma tetraglutamated aminopterin. In some embodiments, theliposome is targeted and unpegylated and the interior space of theliposome contains between 10 to 100,000 molecules of gammatetraglutamated aminopterin, or any range therein between. In furtherembodiments, the liposome is targeted and unpegylated and the interiorspace of the liposome contains between 10,000 to 100,000 molecules ofgamma tetraglutamated aminopterin, or any range therein between. In someembodiments, the liposome is non-targeted and unpegylated (NTLp-γPAMN)and has an interior space that contains less than 500,000 or 200,000molecules of gamma tetraglutamated aminopterin. In some embodiments, theliposome is non-targeted and unpegylated and the interior space of theliposome contains between 10 to 100,000 molecules of gammatetraglutamated aminopterin, or any range therein between. In furtherembodiments, the liposome is non-targeted and unpegylated and theinterior space of the liposome contains between 10,000 to 100,000molecules of gamma tetraglutamated aminopterin, or any range thereinbetween.

In some embodiments, the liposome encapsulates gamma pentaglutamatedaminopterin (i.e., Lp-γPAMN, including PLp-γPAMN, TPLp-γPAMN, TLp-γPAMN,and NTLp-γPAMN) and has an interior space that contains less than500,000 or 200,000 molecules of gamma pentaglutamated aminopterin. Insome embodiments, the liposome interior space contains between 10 to100,000 molecules of gamma pentaglutamated aminopterin, or any rangetherein between. In some embodiments, the liposome interior spacecontains between 10,000 to 100,000 molecules of gamma pentaglutamatedaminopterin, or any range therein between. In some embodiments, theliposome is unpegylated and has an interior space that contains lessthan 500,000 or 200,000 molecules of gamma pentaglutamated aminopterin.In some embodiments, the liposome is unpegylated and the interior spaceof the liposome contains between 10 to 100,000 molecules of gammapentaglutamated aminopterin, or any range therein between. In furtherembodiments, the liposome is unpegylated and the interior space of theliposome contains between 10,000 to 100,000 molecules of gammapentaglutamated aminopterin, or any range therein between. In someembodiments, the liposome is targeted and unpegylated (TLp-γPAMN) andhas an interior space that contains less than 500,000 or 200,000molecules of gamma pentaglutamated aminopterin. In some embodiments, theliposome is targeted and unpegylated and the interior space of theliposome contains between 10 to 100,000 molecules of gammapentaglutamated aminopterin, or any range therein between. In furtherembodiments, the liposome is targeted and unpegylated and the interiorspace of the liposome contains between 10,000 to 100,000 molecules ofgamma pentaglutamated aminopterin, or any range therein between. In someembodiments, the liposome is non-targeted and unpegylated (NTLp-γPAMN)and has an interior space that contains less than 500,000 or 200,000molecules of gamma pentaglutamated aminopterin. In some embodiments, theliposome is non-targeted and unpegylated and the interior space of theliposome contains between 10 to 100,000 molecules of gammapentaglutamated aminopterin, or any range therein between. In furtherembodiments, the liposome is non-targeted and unpegylated and theinterior space of the liposome contains between 10,000 to 100,000molecules of gamma pentaglutamated aminopterin, or any range thereinbetween.

In some embodiments, the liposome encapsulates gamma hexaglutamatedaminopterin (i.e., Lp-γPAMN, including PLp-γPAMN, TPLp-γPAMN, TLp-γPAMN,and NTLp-γPAMN) and has an interior space that contains less than500,000 or 200,000 molecules of gamma hexaglutamated aminopterin. Insome embodiments, the liposome interior space contains between 10 to100,000 molecules of gamma hexaglutamated aminopterin, or any rangetherein between. In further embodiments, the liposome interior spacecontains between 10,000 to 100,000 molecules of gamma hexaglutamatedaminopterin, or any range therein between. In some embodiments, theliposome is unpegylated and has an interior space that contains lessthan 500,000 or 200,000 molecules of gamma hexaglutamated aminopterin.In some embodiments, the liposome is unpegylated and the interior spaceof the liposome contains between 10 to 100,000 molecules of gammahexaglutamated aminopterin, or any range therein between. In furtherembodiments, the liposome is unpegylated and the interior space of theliposome contains between 10,000 to 100,000 molecules of gammahexaglutamated aminopterin, or any range therein between. In someembodiments, the liposome is targeted and unpegylated (TLp-γPAMN) andhas an interior space that contains less than 500,000 or 200,000molecules of gamma hexaglutamated aminopterin. In some embodiments, theliposome is targeted and unpegylated and the interior space of theliposome contains between 10 to 100,000 molecules of gammahexaglutamated aminopterin, or any range therein between. In furtherembodiments, the liposome is targeted and unpegylated and the interiorspace of the liposome contains between 10,000 to 100,000 molecules ofgamma hexaglutamated aminopterin, or any range therein between. In someembodiments, the liposome is non-targeted and unpegylated (NTLp-γPAMN)and has an interior space that contains less than 500,000 or 200,000molecules of gamma hexaglutamated aminopterin. In some embodiments, theliposome is non-targeted and unpegylated and the interior space of theliposome contains between 10 to 100,000 molecules of gammahexaglutamated aminopterin, or any range therein between. In furtherembodiments, the liposome is non-targeted and unpegylated and theinterior space of the liposome contains between 10,000 to 100,000molecules of gamma hexaglutamated aminopterin, or any range thereinbetween.

In some embodiments, the disclosure provides a liposomal gammapolyglutamated aminopterin composition wherein the liposome encapsulatesgamma polyglutamated aminopterin or a salt or acid thereof, and one ormore aqueous pharmaceutically acceptable carriers. In some embodiments,the liposome interior space contains trehalose. In some embodiments, theliposome interior space contains 5% to 20% weight of trehalose. In someembodiments, the liposome interior space contains HBS at a concentrationof between 1 to 200 mM and a pH of between 2 to 8. In some embodiments,liposome interior space has a pH 5-8, or any range therein between. Insome embodiments, liposome interior space has a pH 6-7, or any rangetherein between. In some embodiments, the liposome interior space has atotal concentration of sodium acetate and calcium acetate of between 50mM to 500 mM, or any range therein between.

A Non-Polyglutamated Polyglutamatable Antifolates

In some embodiments, the liposome gamma polyglutamated aminopterin(e.g., Lp-γPAMN, including PLp-γPAMN, TPLp-γPAMN, TLp-γPAMN, andNTLp-γPAMN) compositions comprise gamma polyglutamated aminopterin e.g.,an γPAMN disclosed herein) and one or more non-polyglutamated,polyglutamatable antifolate compositions.

In some embodiments, the Lp-γPAMN (e.g., PLp-γPAMN, TPLp-γPAMN,TLp-γPAMN, and NTLp-γPAMN) comprises gamma polyglutamated aminopterin(e.g., an γPAMN disclosed herein) and aminopterin (AMN). In someembodiments, the Lp-γPAMN (i.e., liposome gamma polyglutamatedaminopterin) comprises gamma polyglutamated aminopterin and apolyglutamatable antifolate selected from the group consisting of:aminopterin, methotrexate (MTX), pemetrexed (PMX), lometrexol (LMX),raltitrexed (RTX), pralatrexate, AG2034, GW1843, and LY309887. In someembodiments, the Lp-γPAMN comprises gamma polyglutamated aminopterin andlometrexol. In some embodiments, the Lp-γPAMN comprises gammapolyglutamated aminopterin and pemetrexed. In some embodiments, theLp-γPAMN comprises gamma polyglutamated aminopterin and leucovorin. Insome embodiments, the Lp-γPAMN comprises gamma polyglutamatedaminopterin and a triazine antifolate derivative (e.g., a sulphonylfluoride triazine such as NSC 127755). In some embodiments, the Lp-γPAMNcomprises gamma polyglutamated aminopterin and a serinehydroxymethyltransferase (SHMT2) inhibitor. In some embodiments, theSHMT2 inhibitor is an antifolate (e.g., a polyglutamatable ornonpolyglutamatable antifolate). In some embodiments, the SHMT2inhibitor is an antifolate.

B Non-Polyglutamatable Antifolates

In some embodiments, the Lp-γPAMN (e.g., PLp-γPAMN, TPLp-γPAMN,TLp-γPAMN, and NTLp-γPAMN) comprises a gamma polyglutamated aminopterin(e.g., an γPAMN disclosed herein) and a so-called “non-polyglutamatable”antifolate. In some embodiments, the liposome comprises a gammapolyglutamated aminopterin and a non-polyglutamatable antifolate thatinhibits one or more enzymes in the folate cycle metabolic pathway. Infurther embodiments, the non-polyglutamatable antifolate inhibits one ormore enzymes selected from: thymidylate synthase (TS), dihydrofolatereductase (DHFR), glycinamide ribonucleotide (GAR) transformylase, andaminoimidazole carboxamide ribonucleotide (AICAR) transformylase. Insome embodiments, the liposome comprises a gamma polyglutamatedaminopterin and a non-polyglutamatable antifolate that inhibits DHFR. Insome embodiments, the liposome comprises a gamma polyglutamatedaminopterin and a non-polyglutamatable antifolate that inhibits TS. Insome embodiments, the liposome comprises a gamma polyglutamatedaminopterin and a non-polyglutamatable antifolate that inhibits GAR orAICAR transformylase. In further embodiments, the non-polyglutamatableantifolate is selected from the group consisting of: trimetrexate (TMQ),piritrexim (BW301U), and talotrexin (PT523). In further embodiments, thenon-polyglutamatable antifolate is selected from the group consistingof: nolatrexed (AG337), plevitrexed (ZD9331, BGC9331), and BGC 945 (ONX0801).

C Platinums

In some embodiments, the liposome comprises a gamma polyglutamatedaminopterin (Lp-γPAMN, such as e.g., PLp-γPAMN, TPLp-γPAMN, TLp-γPAMN,and NTLp-γPAMN) comprises a gamma polyglutamated aminopterin (e.g., anγPAMN disclosed herein) and a platinum-based chemotherapeutic agent or asalt or acid, thereof. In some embodiments, the liposome contains agamma polyglutamated aminopterin/platinum based agent complex (e.g., asdescribed in Section IIC).

In some embodiments, the Lp-γPAMN comprises a platinum-basedchemotherapeutic agent selected from the group consisting of: cisplatin,carboplatin, and oxaliplatin, or a salt or acid thereof. In otherembodiments, the Lp-γPAMN comprises an analog of a platinum-basedchemotherapeutic agent selected from the group consisting of: cisplatin,carboplatin, or oxaliplatin, or a salt or acid thereof.

In some embodiments, the Lp-γPAMN comprises a gamma polyglutamatedaminopterin and cisplatin or a salt or acid thereof. In someembodiments, the Lp-γPAMN comprises a gamma polyglutamated aminopterinand a cisplatin analog, or a salt or acid thereof.

In some embodiments, the Lp-γPAMN comprises a gamma polyglutamatedaminopterin and carboplatin, or a salt or acid thereof. In someembodiments, the liposome comprises a gamma polyglutamated aminopterinand carboplatin analog, or a salt or acid thereof.

In some embodiments, the Lp-γPAMN comprises a gamma polyglutamatedaminopterin and oxaliplatin, or a salt or acid thereof. In someembodiments, the liposome comprises a gamma polyglutamated aminopterinand an oxaliplatin analog, or a salt or acid thereof.

In some embodiments, the liposome comprises a gamma polyglutamatedaminopterin (e.g., an γPAMN disclosed herein) and a platinum-basedchemotherapeutic agent selected from the group consisting of:nedaplatin, heptaplatin, and lobaplatin, nedaplatin, heptaplatin, andlobaplatin or a salt or acid thereof. In some embodiments, the Lp-γPAMNcomprises a gamma polyglutamated aminopterin and an analog of aplatinum-based chemotherapeutic agent selected from the group consistingof: nedaplatin, heptaplatin, and lobaplatin, or a salt or acid thereof.

In some embodiments, the Lp-γPAMN comprises a gamma polyglutamatedaminopterin and a platinum-based chemotherapeutic agent selected fromthe group consisting of: stratoplatin, paraplatin, platinol,cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin,tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamine,traplatin, enloplatin, JM-216, 254-S, NK 121, CI-973, DWA 2114R, NDDP,and dedaplatin, or a salt or acid thereof. In some embodiments, theLp-γPAMN comprises a gamma polyglutamated aminopterin and an analog of aplatinum-based chemotherapeutic agent selected from the group consistingof: stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin,spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin,zeniplatin, platinum-triamine, traplatin, enloplatin, JM-216, 254-S, NK121, CI-973, DWA 2114R, NDDP, and dedaplatin, or a salt or acid thereof.

In some embodiments, the liposome composition comprises liposomes thatfurther contain one or more of an immunostimulatory agent, a detectablemarker and a maleimide disposed on at least one of the PEG and theexterior of the liposome.

D Cyclodextrins

In additional embodiments, the γPAMN liposome comprise a γPAMN (e.g., aγPAMN disclosed herein) and a cyclodextrin (e.g., a cyclodextrin inSection IIC, herein).

In some embodiments, the γPAMN liposome comprises a complex formed by acyclodextrin and a therapeutic agent. In some embodiments, thetherapeutic agent is a cytotoxic compound or a salt or acid thereof. Ina further embodiment, the therapeutic agent is a chemotherapeutic agentor a salt or acid thereof. In another embodiment, the therapeutic agentis a platinum-based drug. In another embodiment, the therapeutic agentis a taxane-based drug. In further embodiments, the therapeutic agent ofthe cyclodextrin/therapeutic agent complex is a member selected from thegroup consisting of: gemcitabine, a gemcitabine-based therapeutic agent,doxorubicin, an antifolate, an antifolate-based chemotherapeutic, or asalt or acid, acid or free base form thereof. In additional embodiments,the molar ratio of cyclodextrin/therapeutic agent in the complex is inthe range 1-10:1. In some embodiments, the molar ratio ofcyclodextrin/therapeutic agent in the complex is 1:1, 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molarratio of cyclodextrin/therapeutic agent in the complex is in the range1:1-20, 1:1-10, or 1:2-8, or any range therein between. In someembodiments, the molar ratio of cyclodextrin/therapeutic agent is: 1:1,1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14,1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50.

In some embodiments, the γPAMN liposome comprises γPAMN and acyclodextrin/platinum-based chemotherapeutic agent complex. In someembodiments, the platinum-based chemotherapeutic agent is selected fromthe group consisting of: cisplatin, carboplatin, and oxaliplatin, or asalt or acid thereof. In other embodiments, thecyclodextrin/platinum-based chemotherapeutic agent complex comprises ananalog of a cisplatin, carboplatin, oxaliplatin, or a salt or acidthereof. In some embodiments, the molar ratio ofcyclodextrin/platinum-based agent in the complex is in the range 1-10:1.In some embodiments, the molar ratio of cyclodextrin/platinum-basedagent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In other embodiments, the molar ratio ofcyclodextrin/platinum-based agent in the complex is in the range 1:1-20,1:1-10, or 1:2-8, or any range therein between. In some embodiments, themolar ratio of cyclodextrin/platinum-based agent is: 1:1, 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:(21-50), or 1:>50.

In some embodiments, the platinum-based chemotherapeutic agent isselected from the group consisting of: cisplatin, carboplatin, andoxaliplatin, or a salt or acid thereof. In other embodiments, thecyclodextrin/platinum-based chemotherapeutic agent complex comprises ananalog of a cisplatin, carboplatin, oxaliplatin, or a salt or acidthereof. In some embodiments, the molar ratio ofcyclodextrin/platinum-based agent in the complex is in the range 1-10:1.In some embodiments, the molar ratio of cyclodextrin/platinum-basedagent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In other embodiments, the molar ratio ofcyclodextrin/platinum-based agent in the complex is in the range 1:1-20,1:1-10, or 1:2-8, or any range therein between. In some embodiments, themolar ratio of cyclodextrin/platinum-based agent is: 1:1, 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additional embodiments, thecyclodextrin//platinum-based agent complex is encapsulated in a liposome(e.g., as described herein or otherwise known in the art).

In further embodiments, the disclosure provides a complex containingcyclodextrin and cisplatin or a salt or acid thereof. In someembodiments, the molar ratio of cyclodextrin/cisplatin (or cisplatinsalt or acid) in the complex is in the range 1-10:1. In someembodiments, the molar ratio of cyclodextrin/cisplatin (or cisplatinsalt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In other embodiments, the molar ratio ofcyclodextrin/cisplatin (or cisplatin salt or acid) in the complex is inthe range 1:1-20, 1:1-10, or 1:2-8, or any range therein between. Insome embodiments, the molar ratio of cyclodextrin/cisplatin (orcisplatin salt or acid) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:(21-50), or 1:>50. In additional embodiments, thecyclodextrin//cisplatin (or cisplatin salt or acid) complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In another embodiment, the disclosure provides a complex containingcyclodextrin and carboplatin or a salt or acid thereof. In someembodiments, the molar ratio of cyclodextrin/carboplatin (or carboplatinsalt or acid) in the complex is in the range 1-10:1. In someembodiments, the molar ratio of cyclodextrin/carboplatin (or carboplatinsalt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In other embodiments, the molar ratio ofcyclodextrin/carboplatin (or carboplatin salt or acid) in the complex isin the range 1:1-20, 1:1-10, or 1:2-8, or any range therein between. Insome embodiments, the molar ratio of cyclodextrin/carboplatin (orcarboplatin salt or acid) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:(21-50), or 1:>50. In additional embodiments, thecyclodextrin/carboplatin (or carboplatin salt or acid) complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In another embodiment, the disclosure provides a complex containingcyclodextrin and oxaliplatin, or a salt or acid thereof. In someembodiments, the molar ratio of cyclodextrin/oxaliplatin (or oxaliplatinsalt or acid) in the complex is in the range 1-10:1. In someembodiments, the molar ratio of cyclodextrin/oxaliplatin (or oxaliplatinsalt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In other embodiments, the molar ratio ofcyclodextrin/oxaliplatin (or oxaliplatin salt or acid) in the complex isin the range 1:1-20, 1:1-10, or 1:2-8, or any range therein between. Insome embodiments, the molar ratio of cyclodextrin/oxaliplatin (oroxaliplatin salt or acid) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:(21-50), or 1:>50. In additional embodiments, thecyclodextrin/oxaliplatin (or oxaliplatin salt or acid) complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In additional embodiments, the disclosure provides a complex comprisingcyclodextrin and a platinum-based chemotherapeutic agent selected fromthe group consisting of: nedaplatin, heptaplatin, lobaplatin,stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin,spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin,zeniplatin, platinum-triamine, traplatin, enloplatin, JM216, NK121,CI973, DWA 2114R, NDDP, and dedaplatin, or a salt or acid thereof. Inother embodiments, the cyclodextrin/platinum-based chemotherapeuticagent complex comprises an analog of nedaplatin, heptaplatin,lobaplatin, stratoplatin, paraplatin, platinol, cycloplatin,dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin,iproplatin, ormaplatin, zeniplatin, platinum-triamine, traplatin,enloplatin, JM216, NK121, CI973, DWA 2114R, NDDP, or dedaplatin, or asalt or acid thereof. In some embodiments, the molar ratio ofcyclodextrin/oxaliplatin (or oxaliplatin salt or acid) in the complex isin the range 1-10:1. In some embodiments, the molar ratio ofcyclodextrin/platinum-based chemotherapeutic agent (or salt or acid oranalog thereof) in the complex 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,(21-50):1, or >50:1. In other embodiments, the molar ratio ofcyclodextrin/platinum-based chemotherapeutic agent (or salt or acid oranalog thereof) in the complex is in the range 1:1-20, 1:1-10, or 1:2-8,or any range therein between. In some embodiments, the molar ratio ofcyclodextrin/platinum-based chemotherapeutic agent (or salt or acid oranalog thereof) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10,1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50),or 1:>50. In additional embodiments, the cyclodextrin/platinum-basedchemotherapeutic agent (or salt or acid or analog thereof) complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In some embodiments, the disclosure provides a composition comprising acyclodextrin/taxane-based chemotherapeutic agent complex. In someembodiments, the taxane-based chemotherapeutic agent is selected fromthe group consisting of: paclitaxel (PTX), docetaxel (DTX), larotaxel(LTX), and cabazitaxel (CTX), or a salt or acid thereof. In someembodiments, the molar ratio of cyclodextrin/taxane-based agent in thecomplex is in the range 1-10:1. In some embodiments, the molar ratio ofcyclodextrin/taxane-based agent in the complex is 1:1, 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molarratio of cyclodextrin/taxane-based agent in the complex is in the range1:1-20, 1:1-10, or 1:2-8, or any range therein between. In someembodiments, the molar ratio of cyclodextrin/taxane-based agent is: 1:1,1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14,1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additionalembodiments, the cyclodextrin/taxane-based agent complex is encapsulatedin a liposome (e.g., as described herein or otherwise known in the art).

In additional embodiments, the disclosure provides a complex comprisingcyclodextrin and paclitaxel (PTX), or a salt or acid thereof. In otherembodiments, the cyclodextrin/taxane-based chemotherapeutic agentcomplex comprises an analog of paclitaxel (PTX), or a salt or acidthereof. In some embodiments, the molar ratio of cyclodextrin/paclitaxel(or paclitaxel salt or acid) in the complex is in the range 1-10:1. Insome embodiments, the molar ratio of cyclodextrin/paclitaxel (orpaclitaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1,19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratioof cyclodextrin/paclitaxel (or paclitaxel salt or acid) in the complexis in the range 1:1-20, 1:1-10, or 1:2-8, or any range therein between.In some embodiments, the molar ratio of cyclodextrin/paclitaxel (orpaclitaxel salt or acid) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:(21-50), or 1:>50. In additional embodiments, thecyclodextrin/paclitaxel (or paclitaxel salt or acid) complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In additional embodiments, the disclosure provides a complex comprisingcyclodextrin and docetaxel (DTX), or a salt or acid thereof. In otherembodiments, the cyclodextrin/taxane-based chemotherapeutic agentcomplex comprises an analog of docetaxel (DTX), or a salt or acidthereof. In some embodiments, the molar ratio of cyclodextrin/docetaxel(or docetaxel salt or acid) in the complex is in the range 1-10:1. Insome embodiments, the molar ratio of cyclodextrin/docetaxel (ordocetaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1,19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratioof cyclodextrin/docetaxel (or docetaxel salt or acid) in the complex isin the range 1:1-20, 1:1-10, or 1:2-8, or any range therein between. Insome embodiments, the molar ratio of cyclodextrin/docetaxel (ordocetaxel salt or acid) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:(21-50), or 1:>50. In additional embodiments, thecyclodextrin/docetaxel (or docetaxel salt or acid) complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In additional embodiments, the disclosure provides a complex comprisingcyclodextrin and larotaxel (LTX), or a salt or acid thereof. In otherembodiments, the cyclodextrin/taxane-based chemotherapeutic agentcomplex comprises an analog of larotaxel (LTX), or a salt or acidthereof. In some embodiments, the molar ratio of cyclodextrin/larotaxel(or larotaxel salt or acid) in the complex is in the range 1-10:1. Insome embodiments, the molar ratio of cyclodextrin/larotaxel (orlarotaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1,19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratioof cyclodextrin/larotaxel (or larotaxel salt or acid) in the complex isin the range 1:1-20, 1:1-10, or 1:2-8, or any range therein between. Insome embodiments, the molar ratio of cyclodextrin/larotaxel (orlarotaxel salt or acid) is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:(21-50), or 1:>50. In additional embodiments, thecyclodextrin/larotaxel (or larotaxel salt or acid) complex isencapsulated in a liposome (e.g., as described herein or otherwise knownin the art).

In additional embodiments, the disclosure provides a complex comprisingcyclodextrin and cabazitaxel (CTX), or a salt or acid thereof. In otherembodiments, the cyclodextrin/taxane-based chemotherapeutic agentcomplex comprises an analog of cabazitaxel (CTX), or a salt or acidthereof. In some embodiments, the molar ratio ofcyclodextrin/cabazitaxel (or cabazitaxel salt or acid) in the complex isin the range 1-10:1. In some embodiments, the molar ratio ofcyclodextrin/cabazitaxel (or cabazitaxel salt or acid) in the complex is1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1,14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In otherembodiments, the molar ratio of cyclodextrin/cabazitaxel (or cabazitaxelsalt or acid) in the complex is in the range 1:1-20, 1:1-10, or 1:2-8,or any range therein between. In some embodiments, the molar ratio ofcyclodextrin/cabazitaxel (or cabazitaxel salt or acid) is: 1:1, 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15,1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In additionalembodiments, the cyclodextrin/cabazitaxel (or cabazitaxel salt or acid)complex is encapsulated in a liposome (e.g., as described herein orotherwise known in the art).

The cyclodextrin of the cyclodextrin/therapeutic agent complex can bederivatized or underivatized. In some embodiments, the cyclodextrin isderivatized. In further embodiments, the cyclodextrin is a derivatizedbeta-cyclodextrin (e.g., a hydroxypropyl beta-cyclodextrin (HP-beta-CD),and a sulfobutyl ether beta-CD (SBE)-beta-cyclodextrin). In someembodiments, the cyclodextrin of the cyclodextrin/therapeutic agentcomplex is a derivatized beta-cyclodextrin comprising: 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more 2-hydroxylpropyl-3-group substitutions of hydroxygroups; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sulfoalkyl ether groupsubstitutions of hydroxy groups. In further embodiments, thecyclodextrin of the cyclodextrin/therapeutic agent complex is aderivatized beta-cyclodextrin comprising: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more sulfobutyl ether group substitutions of hydroxy groups.

In some embodiments, the cyclodextrin of the cyclodextrin/therapeuticagent complex contained in the γPAMN liposome composition is aderivatized cyclodextrin of Formula I:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —H, a straight chain or branchedC₁-C₈-alkylene group, a 2-hydroxylpropyl-3-group; or an optionallysubstituted straight-chain or branched C₁-C₆ group, wherein at least oneof R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ is a straight-chain or branchedC₁-C₈-alkylene group or a 2-hydroxylpropyl-3-group.

In some embodiments, the cyclodextrin of the cyclodextrin/therapeuticagent complex contained in the γPAMN liposome composition is aderivatized cyclodextrin of Formula II:

wherein: n is 4, 5, or 6; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,and R₉ are each, independently, —O— or a —O—(C₂-C₆ alkylene)-SO₃ ⁻group; wherein at least one of R₁ and R₂ is independently a —O—(C₂-C₆alkylene)-SO₃ ⁻ group; and S₁, S₂, S₃, S₄, S₅, S₆, S₇, Ss, and S₉ areeach, independently, a —H or a H or a pharmaceutically acceptablecation. In further embodiments, the wherein the pharmaceuticallyacceptable cation is selected from: an alkali metal such as Li⁺, Na⁺, orK⁺; an alkaline earth metal such as Ca⁺², or Mg⁺², and ammonium ions andamine cations such as the cations of (C1-C6)-alkylamines, piperidine,pyrazine, (C1-C6)-alkanolamine and (C4-C8)-cycloalkanolamine.

In some embodiments, the γPAMN liposome comprises between 100 to 100,000of the cyclodextrin/therapeutic agent complexes.

In some embodiments, a cyclodextrin derivative of the γPAMN/cyclodextrincomplex and/or cyclodextrin/therapeutic agent complex is a cyclodextrindisclosed in U.S. Pat. Nos. 6,133,248, 5,874,418, 6,046,177, 5,376,645,5,134,127, 7,034,013, 6,869,939; and Intl. Appl. Publ. No. WO02005/117911, the contents each of which is herein incorporated byreference in its priority.

In some embodiments, the cyclodextrin derivative of thecyclodextrin/therapeutic agent complex is a sulfoalkyl ethercyclodextrin. In some embodiments, the cyclodextrin derivative ofcomplex is a sulfobutyl ether-3-cyclodextrin such as CAPTISOL® (CyDexPharma. Inc., Lenexa, Kans. Methods for preparing sulfobutylether-3-cyclodextrin and other sulfoalkyl ether cyclodextrins are knownin the art.

In some embodiments, the cyclodextrin derivative of thecyclodextrin/therapeutic agent complex is a compound of Formula III:

wherein R equals:

(e) (H)_(21-x) or (—(CH₂)₄—SO₃Na)x, and x=1.0-10.0, 1.0-5.0, 6.0-7.0, or8.0-10.0;

(f) (H)_(21-x) or (—(CH₂CH(OH)CH₃)x, and x=1.0-10.0, 1.0-5.0, 6.0-7.0,or 8.0-10.0;

(g) (H)_(21-x) or (sulfoalkyl ethers)x, and x=1.0-10.0, 1.0-5.0,6.0-7.0, or 8.0-10.0; or

(h) (H)_(21-x) or (—(CH₂)₄—SO₃Na)x, and x=1.0-10.0, 1.0-5.0, 6.0-7.0, or8.0-10.0.

Additional cyclodextrins and cyclodextrin/platinum-based therapeuticcomplexes that can be contained in the γPAMN liposomes and usedaccording to the disclosed methods is disclosed in U.S. Appl. No.62/583,432, the contents of which is herein incorporated by reference itits entirety.

In some embodiments, the γPAMN liposome comprises a complex of acyclodextrin and a platinum-based chemotherapeutic agent, or a saltthereof. In some embodiments, the platinum-based chemotherapeutic agentis cisplatin or a cisplatin analog. In some embodiments, theplatinum-based chemotherapeutic agent is carboplatin. In additionalembodiments, the liposome composition comprises a platinum-basedchemotherapeutic agent is a member selected from the group consistingof: carboplatin, cisplatin, oxaliplatin, satraplatin, picoplatin,nedaplatin, triplatin, tetraplatin, lipoplatin, lobaplatin, ormaplatin,zeniplatin, platinum-triamine, traplatin, enloplatin, JM-216, 254-S, NK121, CI-973, DWA 2114R, NDDP, and dedaplatin. In some embodiments, theγPAMN liposome comprises between 100 to 100,000 platinum-basedchemotherapeutic agent/CD complexes. In additional embodiments, theliposome composition comprises liposomes that have a diameter in therange of 20 nm to 500 nm or 20 nm to 200 nm, or any range thereinbetween. In some embodiments, liposomes in the composition comprisebetween 100 to 100,000 platinum.

(3) Targeted Liposomes

In some embodiments, the disclosure provides a liposomal gammapolyglutamated aminopterin composition wherein the liposome comprises agamma polyglutamated aminopterin and a targeting moiety attached to oneor both of a PEG and the exterior of the liposome, and wherein thetargeting moiety has a specific affinity for a surface antigen on atarget cell of interest. Such liposomes may generally be referred toherein as “targeted liposomes”, e.g., liposomes including one or moretargeting moieties or biodistribution modifiers on the surface of, orotherwise attached to, the liposomes. The targeting moiety of thetargeted liposomes can be any moiety or agent that is capable ofspecifically binding a desired target (e.g., an antigen target expressedon the surface of a target cell of interest). In one embodiment, thetargeted liposome specifically and preferentially binds to a target onthe surface of a target cell of interest that internalizes the targetedliposome into which the liposome encapsulated gamma polyglutamatedaminopterin (e.g., gamma pentaglutamated AMN or gamma hexaglutamatedAMN) exerts its cytotoxic effect. In further embodiments, the targetcell is a cancer cell, a tumor cell or a metastatic cell. In someembodiments, the targeted liposome is pegylated.

The term “attach” or “attached” refers, for example, to any type ofbonding such as covalent bonding, ionic bonding (e.g., avidin-biotin)bonding by hydrophobic interactions, and bonding via functional groupssuch as maleimide, or linkers such as PEG. For example, a detectablemarker, a steric stabilizer, a liposome, a liposomal component, animmunostimulating agent may be attached to each other directly, by amaleimide functional group, or by a PEG-malemide group.

The composition and origination of the targeting moiety is non-limitingto the scope of this disclosure. In some embodiments, the targetingmoiety attached to the liposome is a polypeptide or peptidomimeticligand. Peptide and peptidomimetic targeting moieties include thosehaving naturally occurring or modified peptides, e.g., D or L peptides;gamma, beta, or glamma peptides; N-methyl peptides; azapeptides;peptides having one or more amide, i.e., peptide, linkages replaced withone or more urea, thiourea, carbamate, or sulfonyl urea linkages; orcyclic peptides. A peptidomimetic is a molecule capable of folding intoa defined three-dimensional structure similar to a natural peptide. Insome embodiments, the peptide or peptidomimetic targeting moiety is 2-50amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50amino acids long

In some embodiments, the targeting moiety polypeptide is at least 40amino acid residues in length. In other embodiments, the targetingmoiety polypeptide is at least 50, 60, 75, 100, 125, 150, 175, 200, 250,or 300 amino acid residues in length.

In additional embodiments, the targeting moiety polypeptide such as anantibody or an antigen-binding antibody fragment that binds a targetantigen with an equilibrium dissociation constant (Kd) in a range of0.5×10⁻¹⁰ to 10×10⁻⁶ as determined using BIACORE analysis.

In some embodiments, the targeting moiety is an antibody or an antibodyderivative. In other embodiments, the binding domain of the targetingmoiety polypeptide is not derived from the antigen binding domain of anantibody. In some embodiments, the targeting moiety is a polypeptidederived from a binding scaffold selected from the group consisting of aDARPin, affilin, and armadillo repeat, D domain (see, e.g., WO2016/164308), Z-domain (Affibody), adnectin, lipocalin, affilin,anticalin, knottin, fynomer, atrimer, kunitz domain (see, e.g., WO2004/063337), CTLA4, or avimer (see, e.g., U.S. Publ. Nos. 2004/0175756,2005/0053973, 2005/0048512, and 2006/0008844).

In additional embodiments, the targeting moiety is an antibody or aderivative of the antigen binding domain of an antibody that hasspecific affinity for an epitope on a cell surface antigen of interestexpressed on the surface of a target cell. In some embodiments, thetargeting moiety is a full-length antibody. In some embodiments, thetargeting moiety is an antigen binding portion of an antibody. In someembodiments, the targeting moiety is an scFv. In other embodiments, thetargeting moiety is a Fab. In some embodiments, the targeting moietycomprises a binding domain derived from the antigen binding domain of anantibody (e.g., an scFv, Fab, Fab′, F(ab′)2, an Fv fragment, adisulfide-linked Fv (sdFv), a Fd fragment containing VH and CH1 domains,an scFv, a minibody, a BiTE, a Tandab, a diabody ((VL-VH)₂ or (VH-VL)₂),a single domain antibody (e.g., an sdAb such as a nanobody (either VL orVH)), and a camelid VHH domain). In some embodiments, the targetingmoiety comprises one or more complementarity determining regions (CDRs)of antibody origin. Examples of suitable antibody-based targetingmoieties for the disclosed targeted liposomes include a full-lengthhuman antibody, a humanized antibody, a chimeric antibody, an antigenbinding fragment of an antibody, a single chain antibody, asingle-domain antibody, a bi-specific antibody, a synthetic antibody, apegylated antibody and a multimeric antibody. The antibody of theprovided targeted liposomes can have a combination of the abovecharacteristics. For example, a humanized antibody can be an antigenbinding fragment and can be pegylated and multimerized as well.

The term “humanized antibody” refers to forms of non-human (e.g.,murine) antibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e.g., mouse, rat, rabbit, and hamster) that have the desiredspecificity, affinity, and capability (Jones et al., Nature 321:522-525(1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,Science 239:1534-1536 (1988)). In some instances, the Fv frameworkregion (FR) residues of a human immunoglobulin are replaced with thecorresponding residues in an antibody from a non-human species that hasthe desired specificity, affinity, and capability. The humanizedantibody can be further modified by the substitution of additionalresidues either in the Fv framework region and/or within the replacednon-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDR regionsthat correspond to the non-human immunoglobulin whereas all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. Nos. 5,225,539 and5,639,641.

In further embodiments, the targeting moiety has specific affinity foran epitope on a surface antigen of a target cell of interest. In someembodiments, the target cell is a cancer cell. In some embodiments, thetarget cell is a tumor cell. In other embodiments, the target cell is animmune cell.

In some embodiments, the targeting moiety has specific affinity for anepitope expressed on a tumor cell surface antigen. The term “tumor cellsurface antigen” refers to an antigen that is common to a specifichyperproliferative disorder such as cancer. In some embodiments, thetargeting moiety has specific affinity for an epitope of a tumor cellsurface antigen that is a tumor associated antigen (TAA). A TAA is anantigen that is found on both tumor and some normal cells. A TAA may beexpressed on normal cells during fetal development when the immunesystem is immature and unable to respond or may be normally present atextremely low levels on normal cells but which are expressed at muchhigher levels on tumor cells. Because of the dynamic nature of tumors,in some instances, tumor cells may express unique antigens at certainstages, and at others also express antigens that are also expressed onnon-tumor cells. Thus, inclusion of a certain marker as a TAA does notpreclude it being considered a tumor specific antigen. In someembodiments, the targeting moiety has specific affinity for an epitopeof a tumor cell surface antigen that is a tumor specific antigen (TSA).A TSA is an antigen that is unique to tumor cells and does not occur onother cells in the body. In some embodiments, the targeting moiety hasspecific affinity for an epitope of a tumor cell surface antigenexpressed on the surface of a cancer including but not limited toprimary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer(e.g., NSCLC or SCLC), liver cancer, non-Hodgkin's lymphoma, Hodgkin'slymphoma, leukemias, multiple myeloma, glioblastoma, neuroblastoma,uterine cancer, cervical cancer, renal cancer, thyroid cancer, bladdercancer, kidney cancer, mesothelioma, and adenocarcinomas such as breastcancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancerand other cancers known in the art In some embodiments, the targetingmoiety has specific affinity for an epitope of a cell surface antigenexpressed on the surface of a cell in the tumor microenvironment (e.g.,and antigen such as VEGFR and TIE1, or TIE2 expressed on endothelialcells and macrophage, respectively, or an antigen expressed on tumorstromal cells such as cancer-associated fibroblasts (CAFs) tumorinfiltrating T cells and other leukocytes, and myeloid cells includingmast cells, eosinophils, and tumor-associated macrophages (TAM).

In some embodiments, the targeted liposome γPAMN composition (e.g.,TLp-γPAMN or TPLp-γPAMN) comprises a targeting moiety that has specificaffinity for an epitope of a cancer or tumor cell surface antigen thatis preferentially/differentially expressed on a target cell such as acancer cell or tumor cell, compared to normal or non-tumor cells, thatis present on a tumor cell but absent or inaccessible on a non-tumorcell. For example, in some situations, the tumor antigen is on thesurface of both normal cells and malignant cancer cells but the tumorepitope is only exposed in a cancer cell. As a further example, a tumorcell surface antigen may experience a confirmation change in a cancerousstate that causes a cancer cell specific epitope to be present. Atargeting moiety with specific affinity to an epitope on a targetabletumor cell surface antigen described herein or otherwise known in theart is useful and is encompassed by the disclosed compositions andmethods. In some embodiments, the tumor cell with the tumor cell surfaceantigen is a cancer cell. Examples of such tumor cell surface antigensinclude, without limitation folate receptor alpha, folate receptor betaand folate receptor delta.

In further embodiments, the targeting moiety comprises a polypeptidetargeting moiety such as an antibody or an antigen-binding antibodyfragment and the targeting moiety has binding specificity for a folatereceptor. In some embodiments, the targeting moiety binds a folatereceptor with an equilibrium dissociation constant (Kd) in a range of0.5×10⁻¹⁰ to 10×10⁻⁶ as determined using BIACORE analysis. In someembodiments, the folate receptor bound by the targeting moiety is one ormore folate receptors selected from the group consisting of: folatereceptor alpha (FR-α), folate receptor beta (FR-β), and folate receptordelta (FR-δ). In a further embodiment, the targeting moiety has specificaffinity for at least two antigens selected from the group consisting offolate receptor alpha, folate receptor beta, and folate receptor delta.In another embodiment, the targeting moiety has specific affinity forfolate receptor alpha; folate receptor beta; and folate receptor delta.

In some embodiments, the targeting moiety has a specific affinity for anepitope of a cell surface antigen that internalizes the targeting moietyupon binding. Numerous cell surface antigens that internalize bindingpartners such as antibodies upon binding are known in the art and areenvisioned to be binding targets for the targeting moieties expressed onthe targeted liposome γPAMN compositions (e.g., TLp-γPAMN or TPLp-γPAMN)disclosed herein.

In some embodiments, the targeting moiety has a specific affinity for anepitope of a cell surface antigen selected from the group consisting of:GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin,Fibronectin Extra-domain B (ED-B), VEGFR2 (CD309), Tenascin, CollagenIV, Periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, the targetingmoiety has a specific affinity for an epitope on a cell surfaceantigen(s) derived from, or determined to be expressed on, a specificsubject's cancer (tumor) such as a neoantigen.

In some embodiments, the targeting moiety has a specific affinity for anepitope of a cell surface antigen selected from the group consisting ofmannose-6-phosphate receptor, transferrin receptor, and a cell adhesionmolecule (CAM). In further embodiments, the targeting moiety has aspecific affinity for an epitope of a CAM is selected from the groupconsist of: intercellular adhesion molecule (ICAM), platelet-endothelialcell adhesion molecule (PECAM), activated leukocyte cell adhesionmolecule (ALCAM), B-lymphocyte cell adhesion molecule (BL-CAM), vascularcell adhesion molecule (VCAM), mucosal vascular addressin cell adhesionmolecule (MAdCAM), CD44, LFA-2, LFA-3, and basigin.

A discussed herein, folate receptors (FRs) are distinct from reducedfolate carriers (RFCs) and exploit different pathways for bringingfolates and antifolates into cells. In some embodiments, the targetingmoiety specifically binds a folate receptor. In further embodiments, thetargeting moiety specifically binds a folate receptor selected fromfolate receptor alpha, folate receptor beta and folate receptor delta.Antibodies to folate receptor alpha can routinely be generated usingtechniques known in the art. Moreover, the sequences of numerousanti-folate receptor antibodies are in the public domain and/orcommercially available and are readily obtainable.

Murine antibodies against folate receptor are examples of antibodiesthat can be used as targeting moieties of the disclosed targetedliposome is a murine antibody against folate receptor. The sequence ofthese antibodies are known and are described, for example, in U.S. Pat.Nos. 5,646,253; 8,388,972; 8,871,206; and 9,133,275, and Intl. Appl.Nos. PCT/US2011/056966, and PCT/US2012/046672. For example, based on thesequences already in the public domain, the gene for the antibodies canbe synthesized and placed into a transient expression vector and theantibody was produced in HEK-293 transient expression system. Theantibody can be a complete antibody, a Fab, or any of the variousantibody variations discussed herein or otherwise known in the art.

In some embodiments, the targeted liposome (e.g., TL-γPAMN or TPL-γPAMN)contains from 1 to 1,000, 30-1,000, 50-1,000, or more than 1,000,targeting moieties on its surface. In some embodiments, the targetedliposome contains from 30 to 500, 30 to 250 or 30-200, targetingmoieties, or any range therein between. In some embodiments, thetargeted liposome contains less than 220 targeting moieties, less than200 targeting moieties, or less than 175 targeting moieties. In someembodiments, the targeting moiety is non-covalently bonded to theoutside of the liposome (e.g., via ionic interaction or a GPI anchor).

In some embodiments, the molecules on the outside of the targetedliposome (e.g., TL-γPAMN or TPL-γPAMN) include a lipid, a targetingmoiety, a steric stabilizer (e.g., a PEG), a maleimide, and acholesterol. In some embodiments, the targeting moiety is covalentlybound via a maleimide functional group. In some embodiments, thetargeting moiety is covalently bound to a liposomal component or asteric stabilizer such as a PEG molecule. In some embodiments, all thetargeting moieties of the liposome are bound to one component of theliposome such as a PEG. In other embodiments, the targeting moieties ofthe targeted liposome are bound to different components of the liposome.For example, some targeting moieties may be bound to the lipidcomponents or cholesterol, some targeting moieties may be bound to thesteric stabilizer (e.g., PEG) and still other targeting moieties may bebound to a detectable marker or to another targeting moiety. In someembodiments, the outside of the targeted liposome (e.g., TL-γPAMN orTPL-γPAMN) further comprises one or more of an immunostimulatory agent,a detectable marker and a maleimide disposed on at least one of the PEGand the exterior of the liposome.

In some embodiments, the targeted liposome (e.g., TL-γPAMN or TPL-γPAMN)is anionic or neutral. In some embodiments, the targeted anionic orneutral liposome has a diameter in the range of 20 nm to 500 nm or 20 nmto 200 nm, or any range therein between. In further embodiments, thetargeted anionic or neutral liposome has a diameter in the range of 80nm to 120 nm, or any range therein between.

In other embodiments, the targeted liposome (e.g., TL-γPAMN orTPL-γPAMN) is cationic. In some embodiments, the targeted anionic orneutral liposome has a diameter in the range of 20 nm to 500 nm or 20 nmto 200 nm, or any range therein between. In further embodiments, thetargeted anionic or neutral liposome has a diameter in the range of 80nm to 120 nm, or any range therein between.

In additional embodiments, the liposomal composition comprises targetedliposomes (e.g., TL-γPAMN or TPL-γPAMN) and 30-70%, 30-60%, or 30-50%,w/w of the gamma polyglutamated AMN, or any range therein between. Insome embodiments, the targeted liposomes comprise at least 1%, 5%, 10%,15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or morethan 75%, w/w of the alpha polyglutamated AMN. In some embodiments,during the process of preparing the targeted liposomes, at least 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, ormore than 75%, of the starting material of gamma polyglutamated AMN isencapsulated (entrapped) in the targeted liposomes.

In some embodiments, the targeted liposomal compositions comprise30-70%, 30-60%, or 30-50%, w/w of the gamma tetraglutamated AMN, or anyrange therein between In some embodiments, the targeted liposomescomprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the gammatetraglutamated AMN. In some embodiments, during the process ofpreparing the targeted liposomes, at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, ofthe starting material of gamma tetraglutamated AMN is encapsulated(entrapped) in the targeted liposomes.

In some embodiments, the targeted liposomal compositions comprise30-70%, 30-60%, or 30-50%, w/w of the gamma pentaglutamated AMN, or anyrange therein between In some embodiments, the targeted liposomescomprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the gammapentaglutamated AMN. In some embodiments, during the process ofpreparing the targeted liposomes, at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, ofthe starting material of gamma pentaglutamated AMN is encapsulated(entrapped) in the targeted liposomes.

In some embodiments, the targeted liposomal compositions comprise30-70%, 30-60%, or 30-50%, w/w of the gamma hexaglutamated AMN, or anyrange therein between In some embodiments, the targeted liposomescomprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the gammahexaglutamated AMN. In some embodiments, during the process of preparingthe targeted liposomes, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, of thestarting material of gamma hexaglutamated AMN is encapsulated(entrapped) in the targeted liposomes.

Methods and techniques for covalently associating polypeptide targetingmoieties with a liposome surface molecule are known in the art and canreadily be applied to prepare the TL-γPAMN or TPL-γPAMN liposomecompositions.

Chemical binding of non-proteinaceous targeting moieties and othercompositions to the liposomal surface may be employed. Thus, anon-proteinaceous moiety, may be covalently or non-covalently linked to,embedded or adsorbed onto the liposome using any linking or bindingmethod and/or any suitable chemical linker known in the art. The exacttype and chemical nature of such cross-linkers and cross linking methodsis preferably adapted to the type of affinity group used and the natureof the liposome. Methods for binding or adsorbing or linking thetargeting moiety are also well known in the art. For example, in someembodiments, the targeting moiety may be attached to a group at theinterface via, but not limited to, polar groups such as amino, SH,hydroxyl, aldehyde, formyl, carboxyl, His-tag or other polypeptides. Inaddition, the targeting moiety may be attached via, but not limited to,active groups such as succinimidyl succinate, cyanuric chloride, tosylactivated groups, imidazole groups, CNBr, NHS, Activated CH, ECH, EAH,Epoxy, Thiopropyl, Activated Thiol, etc., Moreover, the targeting moietymay be attached via, but not limited to, hydrophobic bonds (Van DerWaals) or electrostatic interactions that may or may not includecross-linking agents (e.g., bivalent anions, poly-anions, poly-cationsetc.).

(4) Manufacture of Liposomes

In some embodiments, the disclosure provides a method of making aliposomal composition disclosed herein. In one embodiment, the methodincludes forming a mixture comprising: (1) a liposomal component; and(2) a gamma polyglutamated (e.g., pentaglutamated or hexaglutamated)aminopterin in aqueous solution. In further embodiments, the mixturecomprises a pegylated liposomal component. The mixture is thenhomogenized to form liposomes in the aqueous solution. Further, themixture can be extruded through a membrane to form liposomes enclosingthe gamma polyglutamated aminopterin in an aqueous solution. It isunderstood the liposomal components of this disclosure can comprise anylipid (including cholesterol) including functionalized lipids and lipidsattached to targeting moieties, detectable labels, and stericstabilizers, or any subset of all of these. It is further noted that thebioactive gamma polyglutamated aminopterin in aqueous solution cancomprise any reagents and chemicals discussed herein or otherwise knownin the art for the interior or exterior of the liposome including, forexample, buffers, salts, and cryoprotectants.

In some embodiments, the disclosure provides a method of making atargeted pegylated liposomal gamma polyglutamated aminopterin(targeted-PLp-γPAMN) or non-targeted PLp-γPAMN disclosed herein. In oneembodiment, the method includes forming a mixture comprising: (1) aliposomal component; (2) a gamma polyglutamated (e.g., pentaglutamatedor hexaglutamated) aminopterin in aqueous solution; and (3) thetargeting moiety. The mixture is then homogenized to form liposomes inthe aqueous solution. Further, the mixture may be extruded through amembrane to form liposomes enclosing the targeted gamma polyglutamatedaminopterin in an aqueous solution. It is understood that the targetedpegylated liposomal components can comprise any lipid (includingcholesterol) including functionalized lipids and lipids attached totargeting moieties, detectable labels, and steric stabilizers, or anysubset of all of these. It is further noted that the targeted pegylatedliposome can comprise any reagents and chemicals discussed herein orotherwise known in the art for the interior or exterior of the liposomeincluding, for example, buffers, salts, and cryoprotectants.

The above methods optionally further comprise the step of lyophilizingthe composition after the removing step to form a lyophilizedcomposition. As stated above, targeted-PTPLA or non-targeted-PTPLA inaqueous solution may comprise a cryoprotectant described herein orotherwise known in the art. If the composition is to be lyophilized, acryoprotectant may be preferred.

Additionally, after the lyophilizing step, the method optionally furthercomprises the step of reconstituting the lyophilized composition bydissolving the composition in a solvent after the lyophilizing step.Methods of reconstitution are known in the art. One preferred solvent iswater. Other preferred solvents include saline solutions and bufferedsolutions.

While certain exemplary embodiments, are discussed herein, it isunderstood that liposomes can be made by any method that is known in theart. See, for example, G. Gregoriadis (editor), Liposome Technology,vol. 1-3, 1st edition, 1983; 2nd edition, 1993, CRC Press, 45 BocaRaton, Fla. Examples of methods suitable for making liposomecompositions include extrusion, reverse phase evaporation, sonication,solvent (e.g., ethanol) injection, microfluidization, detergentdialysis, ether injection, and dehydration/rehydration. The size ofliposomes can routinely be controlled by controlling the pore size ofmembranes used for low pressure extrusions or the pressure and number ofpasses utilized in microfluidization or any other suitable methods knownin the art.

In general, the gamma polyglutamated aminopterin is contained inside,that is, in the inner (interior) space of the liposomes. In oneembodiment, substituted ammonium is partially or substantiallycompletely removed from the outer medium surrounding the liposomes. Suchremoval can be accomplished by any suitable means known in the art(e.g., dilution, ion exchange chromatography, size exclusionchromatography, dialysis, ultrafiltration, and precipitation).Accordingly, the methods of making liposomal compositions set forthabove or otherwise known in the art can optionally further comprise thestep of removing gamma polyglutamated aminopterin in aqueous solutionoutside of the liposomes after the extruding step.

In other embodiments, the disclosure provides a targeted pegylatedliposomal gamma polyglutamated aminopterin (TPLp-γPAMN) that selectivelytargets folate receptors comprising: a liposome including an interiorspace, a gamma polyglutamated aminopterin disposed within the interiorspace, a steric stabilizer molecule attached to an exterior of theliposome, and a targeting moiety comprising a protein with specificaffinity for at least one folate receptor, said targeting moietyattached to at least one of the steric stabilizer and the exterior ofthe liposome. The components of this embodiment, may be the same asdescribed for other embodiments, of this disclosure. For example, thetargeted pegylated liposomal gamma polyglutamated aminopterin and thesteric stabilizer which may be PEG, are as described in other parts ofthis disclosure.

In some embodiments, the disclosure provides a method of preparing atargeted composition comprising a pegylated liposome including anentrapped and/or encapsulated gamma polyglutamated aminopterin; atargeting moiety an amino acid chain, the amino acid chain comprising aplurality of amino acids, the targeting moiety having a specificaffinity for at least one type of folate receptor, the specific affinitybeing defined to include an equilibrium dissociation constant (Kd) in arange of 0.5×10⁻¹⁰ to 10×10⁻⁶ moles [0.05 nM to 10 μM] for at least onetype folate receptor, the targeting moiety attached to one or both of aPEG and an exterior of the liposome, the method comprising: forming amixture comprising: liposomal components and gamma polyglutamatedaminopterin in solution; homogenizing the mixture to form liposomes inthe solution; processing the mixture to form liposomes entrapping and/orencapsulating gamma polyglutamated aminopterin; and providing thetargeting moiety on a surface of the liposomes entrapping and/orencapsulating the gamma polyglutamated aminopterin, the targeting moietyhaving specific affinity for at least one of folate receptor alpha(FR-α), folate receptor beta (FR-β) and folate receptor delta (FR-δ). Insome embodiments, the method comprising: forming a mixture comprising:liposomal components and gamma polyglutamated aminopterin in solution;forming liposomes entrapping and/or encapsulating gamma polyglutamatedaminopterin, for example by homogenizing or otherwise processing themixture to form liposomes; and providing a targeting moiety on a surfaceof the liposomes entrapping and/or encapsulating the gammapolyglutamated aminopterin, the targeting moiety having specificaffinity for at least one of folate receptor alpha (FR-α), folatereceptor beta (FR-β) and folate receptor delta (FR-δ). In someembodiments, the processing includes one or more of: thin filmhydration, extrusion, in-line mixing, ethanol injection technique,freezing-and-thawing technique, reverse-phase evaporation, dynamic highpressure microfluidization, microfluidic mixing, double emulsion,freeze-dried double emulsion, 3D printing, membrane contactor method,and stirring, and once the particles have been formed, the particles canhave their sizes further modified by one or more of extrusion andsonication. In some embodiments, during the process of preparing theliposomes at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, or more than 75%, of the starting material ofgamma polyglutamated AMN is encapsulated (entrapped) in the targetedliposomes. In some embodiments, the liposomes are anionic or neutral. Insome embodiments, the targeting moiety has the specific affinity for oneor more of: folate receptor alpha (FR-α), folate receptor beta (FR-β)and folate receptor delta (FR-δ). In further embodiments, the targetingmoiety has the specific affinity for folate receptor alpha (FR-α) andfolate receptor beta (FR-β). In additional embodiments, the targetingmoiety has the specific affinity for an epitope on a tumor cell surfaceantigen that is present on a tumor cell but absent or inaccessible on anon-tumor cell.

B. Antibody Delivery Vehicles

In additional embodiments, the disclosure provides an antibody deliveryvehicle (e.g., ADC). In some embodiments, the disclosure provides animmunoconjugate having the formula (A)-(L)-(γPAMN), wherein: (A) is anantibody or antigen binding fragment of an antibody; (L) is a linker;and (γPAMN) is a γPAMN composition described herein; and wherein saidlinker (L) links (A) to (γPAMN).

In some embodiments, the antibody or antigen binding antibody fragmenthas specific affinity for an epitope of a cell surface antigen on a cellof interest (e.g., an epitope and/or antigen described herein). Incertain embodiments, the antibody binds to an antigen target that isexpressed in or on the cell membrane (e.g., on the cell surface) of acancer/tumor and the antibody is internalized by the cell after bindingto the (antigen) target, after which the γPAMN is releasedintracellularly. In some embodiments, the antibody is a full lengthantibody.

The antibody or antigen binding antibody fragment of the (A)-(L)-(γPAMN)immunoconjugate can be an IgA, IgD, IgE, IgG or IgM antibody. Thedifferent classes of immunoglobulins have different and well knownsubunit structures and three-dimensional configurations. In certainembodiments, the antibody is an IgG antibody. In some embodiments, theantibody is an IgG1, IgG2, IgG3 or IgG4 antibody. In certainembodiments, the antibody is an IgG1 antibody.

In some embodiments, (A) is an antigen binding fragment of an antibody.In some embodiments, (A) is an antigen binding fragment of an antibody.

A “linker” is any chemical moiety that is capable of linking a compound,usually a drug, such as a γPAMN, to an antibody or antigen bindingfragment of an antibody in a stable, covalent manner. The linkers can besusceptible to or be substantially resistant to acid-induced cleavage,light-induced cleavage, peptidase-induced cleavage, esterase-inducedcleavage, and disulfide bond cleavage, at conditions under which thecompound or the antibody remains active. Suitable linkers are known inthe art and include, for example, disulfide groups, thioether groups,acid labile groups, photolabile groups, peptidase labile groups andesterase labile groups. Linkers also include charged linkers, andhydrophilic forms thereof.

In some embodiments, the linker is selected from the group consisting ofa cleavable linker, a non-cleavable linker, a hydrophilic linker, and adicarboxylic acid based linker. In another embodiment, the linker is anon-cleavable linker. In another embodiment, the linker is selected fromthe group consisting: N-succinimidyl 4-(2-pyridyldithio) pentanoate(SPP); N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) orN-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB);N-succinimidyl 4-(maleimidomethyl) cyclohexane-carboxylate (SMCC);N-sulfosuccinimidyl 4-(maleimidomethyl) cyclohex-anecarboxylate(sulfoSMCC); N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB); andN-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester(NHS-PEG4-ma-leimide). In a further embodiment, the linker isN-succinimidyl-[(N-maleimido-propionamido)-tetraethyleneglycol]ester(NHS-PEG4-maleimide).

In some embodiments, the γ polyglutamated AMN is attached (coupled) tothe antibody or antigen binding antibody fragment of the immunoconjugatedirectly, or through a linker using techniques known in the art. Suchattachment of one or more γPAMN can include many chemical mechanisms,such as covalent binding, affinity binding, intercalation, coordinatebinding and complexation. Covalent binding of the γPAMN and antibody orantigen binding antibody fragment can be achieved by direct condensationof existing side chains or by the incorporation of external bridgingmolecules. Many bivalent or polyvalent agents are useful in associatingpolypeptides to other proteins with coupling agents such ascarbodiimides, diisocyanates, glutaraldehyde, diazobenzenes, andhexamethylene diamines. This list is not intended to be exhaustive ofthe various coupling agents known in the art but, rather, is exemplaryof the more common coupling agents. In some embodiments, the antibody orantigen binding antibody fragment is derivatized and then attached tothe γ polyglutamated AMN. Alternatively, the γPAMN can be derivatizedand attached to the antibody or antigen binding antibody fragment usingtechniques known in the art.

In some embodiments, the immunoconjugate comprises an antibody or anantigen-binding fragment of an antibody and γPAMN containing 4, 5, 2-10,4-6, or more than 5, glutamyl groups (including the glutamyl group inaminopterin). In some embodiments, the immunoconjugate comprises gammapolyglutamated aminopterin that comprises two or more glutamyl groups inthe L-form. In other embodiments, the immunoconjugate comprises gammapolyglutamated aminopterin that comprises a glutamyl group in theD-form. In further embodiments, the immunoconjugate comprises gammapolyglutamated aminopterin that comprises a glutamyl group in the D-formand two or more glutamyl groups in the L-form. In additionalembodiments, the immunoconjugate comprises gamma polyglutamatedaminopterin that comprises two or more glutamyl groups that have aglamma carboxyl linkage. In some embodiments, the immunoconjugatecomprises γpentaglutamated AMN. In further embodiments, theimmunoconjugate comprises L-γpentaglutamated AMN, a D-γpentaglutamatedAMN, or an L- and D-γpentaglutamated AMN. In some embodiments, theimmunoconjugate comprises a γhexaglutamated AMN (Lp-γPAMN). In furtherembodiments, the immunoconjugate comprises an L-γhexaglutamated AMN, aD-γhexaglutamated AMN, or an L- and D-γhexaglutamated AMN.

In some embodiments, the antibody delivery vehicle composition comprisesa gamma polyglutamated aminopterin and an antibody or an antigen bindingantibody fragment that has specific affinity for an epitope on a cellsurface antigen selected from the group consisting of: GONMB, TACSTD2(TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-γ,folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1,mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b,CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16,Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin, FibronectinExtra-domain B (ED-B), VEGFR2 (CD309), Tenascin, Collagen IV, Periostin,endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2,FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7,FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15,CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38,CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138,cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA1,EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3,EphB4, EphB6, an integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA,c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB,TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the delivery vehicle comprises a targeting moietythat has specific affinity for an epitope of a cell surface antigen(s)derived from, or determined to be expressed on, a specific subject'scancer (tumor) such as a neoantigen.

In some embodiments, the antibody delivery vehicle composition comprisesa gamma polyglutamated aminopterin and an antibody or an antigen bindingantibody fragment that has specific affinity for an epitope on anantigen selected from the group consisting of mannose-6-phosphatereceptor, transferrin receptor, and a cell adhesion molecule (CAM). Infurther embodiments, the targeting moiety has a specific affinity for anepitope of a CAM is selected from the group consist of: intercellularadhesion molecule (ICAM), platelet-endothelial cell adhesion molecule(PECAM), activated leukocyte cell adhesion molecule (ALCAM),B-lymphocyte cell adhesion molecule (BL-CAM), vascular cell adhesionmolecule (VCAM), mucosal vascular addressin cell adhesion molecule(MAdCAM), CD44, LFA-2, LFA-3, and basigin

In some embodiments, the antibody delivery vehicle composition comprises1, 2, 3, 4, 5, 5-10, or greater than 10 γ polyglutamated AMN. In someembodiments, the antibody delivery vehicle composition comprises 1, 2,3, 4, 5, 5-10, or greater than 10, γ pentaglutamated AMN. In someembodiments, the antibody delivery vehicle composition comprises 1, 2,3, 4, 5, 5-10, or greater than 10, γ hexaglutamated AMN.

IV. Pharmaceutical Compositions and Administration

In some embodiments, the liposome composition is provided as apharmaceutical composition containing the liposome and a carrier, e.g.,a pharmaceutically acceptable carrier. Examples of pharmaceuticallyacceptable carriers contained in the provided pharmaceuticalcompositions include normal saline, isotonic dextrose, isotonic sucrose,Ringer's solution, and Hanks' solution. In some embodiments, a buffersubstance is added to maintain an optimal pH for storage stability ofthe pharmaceutical composition. In some embodiments, the pH of thepharmaceutical composition is between 6.0 and 7.5. In some embodiments,the pH is between 6.3 and 7.0. In further embodiments, the pH is 6.5.Ideally the pH of the pharmaceutical composition allows for bothstability of liposome membrane lipids and retention of the entrappedentities. Histidine, hydroxyethylpiperazine-ethylsulfonate (HEPES),morpholipoethylsulfonate (MES), succinate, tartrate, and citrate,typically at 2-20 mM concentration, are exemplary buffer substances.Other suitable carriers include, e.g., water, buffered aqueous solution,0.4% NaCl, and 0.3% glycine. Protein, carbohydrate, or polymericstabilizers and tonicity adjusters can be added, e.g., gelatin, albumin,dextran, or polyvinylpyrrolidone. The tonicity of the composition can beadjusted to the physiological level of 0.25-0.35 mol/kg with glucose ora more inert compound such as lactose, sucrose, mannitol, or dextrin.These compositions can routinely be sterilized using conventional,sterilization techniques known in the art (e.g., by filtration). Theresulting aqueous solutions can be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile aqueous medium prior to administration.

The provided pharmaceutical liposome compositions can also contain otherpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, and tonicity adjusting agents, for example, sodium acetate,sodium lactate, sodium chloride, potassium chloride, and calciumchloride. Additionally, the liposome suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as gamma-tocopherol and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

The liposome concentration in the provided fluid pharmaceuticalformulations can vary widely depending upon need, e.g., from less thanabout 0.05% usually or at least about 2-10% to as much as 30-50% byweight and will be selected primarily by fluid volumes, and viscosities,in accordance with the particular mode of administration selected. Forexample, the concentration may be increased to lower the fluid loadassociated with treatment. This may be particularly desirable inpatients having atherosclerosis-associated congestive heart failure orsevere hypertension. Alternatively, liposome pharmaceutical compositionscomposed of irritating lipids may be diluted to low concentrations tolessen inflammation at the site of administration.

Some embodiments, relate to a method of delivering a targeted pegylatedliposomal formulation of gamma polyglutamated aminopterin, to a tumorexpressing folate receptor on its surface. An exemplary method comprisesthe step of administering a liposome pharmaceutical composition providedherein in an amount to deliver a therapeutically effective dose of thetargeted pegylated liposomal gamma polyglutamated aminopterin to thetumor.

The amount of liposome pharmaceutical composition administered willdepend upon the particular gamma polyglutamated aminopterin entrappedinside the liposomes, the disease state being treated, the type ofliposomes being used, and the judgment of the clinician. Generally theamount of liposome pharmaceutical composition administered will besufficient to deliver a therapeutically effective dose of the particulartherapeutic entity.

The quantity of liposome pharmaceutical composition necessary to delivera therapeutically effective dose can be determined by routine in vitroand in vivo methods, common in the art of drug testing. See, forexample, D. B. Budman, A. H. Calvert, E. K. Rowinsky (editors). Handbookof Anticancer Drug Development, L W W, 2003. Therapeutically effectivedosages for various therapeutic compositions are known to those skilledin the art. In some embodiments, a therapeutic entity delivered via thepharmaceutical liposome composition and provides at least the same orhigher activity than the activity obtained by administering the sameamount of the therapeutic entity in its routine non-liposomeformulation. Typically, the dosages for the liposome pharmaceuticalcomposition is in a range for example, between about 0.005 and about5000 mg of the therapeutic entity per square meter (m²) of body surfacearea, most often, between about 0.1 and about 100 mg therapeutic entityper m² of body surface area.

For example, if the subject has a tumor, an effective amount may be thatamount of the agent (e.g., gamma polyglutamated aminopterin composition)that reduces the tumor volume or load (as for example determined byimaging the tumor). Effective amounts can also routinely be assessed bythe presence and/or frequency of cancer cells in the blood or other bodyfluid or tissue (e.g., a biopsy). If the tumor is impacting the normalfunctioning of a tissue or organ, then the effective amount canroutinely be assessed by measuring the normal functioning of the tissueor organ. In some instances the effective amount is the amount requiredto lessen or eliminate one or more, and preferably all, symptoms.

Pharmaceutical compositions comprising the gamma polyglutamatedaminopterin compositions (e.g., liposomes containing a pentaglutamatedor hexaglutamated aminopterin) are also provided. Pharmaceuticalcompositions are sterile compositions that comprise a sample liposomeand preferably gamma polyglutamated aminopterin, preferably in apharmaceutically-acceptable carrier.

Unless otherwise stated herein, a variety of administration routes areavailable. The particular mode selected will depend, upon the particularactive agent selected, the particular condition being treated and thedosage required for therapeutic efficacy. The provided methods can bepracticed using any known mode of administration that is medicallyacceptable and in accordance with good medical practice. In someembodiments, the administration route is an injection. In furtherembodiments, the injection is by a parenteral route elected from anintramuscular, subcutaneous, intravenous, intraarterial,intraperitoneal, intraarticular, intraepidural, intrathecal,intravenous, intramuscular, or intra sternal injection. In someembodiments, the administration route is an infusion. In additionalembodiments, the administration route is oral, nasal, mucosal,sublingual, intratracheal, ophthalmic, rectal, vaginal, ocular, topical,transdermal, pulmonary, or inhalation.

Therapeutic compositions containing γPAMN compositions such as theliposomal γPAMN compositions described herein can be conventionallyadministered intravenously, as by injection of a unit dose, for example.The term “unit dose” when used in reference to a therapeutic compositionprovided herein refers to physically discrete units suitable as unitarydosage for the subject, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect inassociation with the required diluent; e.g., carrier, or vehicle. In aspecific embodiment, therapeutic compositions containing an Adapter areadministered subcutaneously.

In some embodiments, the γ-PAMN composition is administered in a mannercompatible with the dosage formulation, and in a therapeuticallyeffective amount. The quantity to be administered depends on the subjectto be treated, capacity of the subject's system to utilize the activeingredient, and degree of therapeutic effect desired. Precise amounts ofactive ingredient required to be administered depend on the judgment ofthe practitioner and are peculiar to each individual. However, suitabledosage ranges for systemic application are disclosed herein and dependon the route of administration. Suitable regimes for administration arealso variable, but are typified by an initial administration followed byrepeated doses at one or more hour intervals by a subsequent injectionor other administration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in vivo therapies are contemplated.

The γPAMN composition are formulated, dosed, and administered in afashion consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular patient being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Thedosage ranges for the administration of γPAMN composition are thoselarge enough to produce the desired effect in which the disease symptomsmediated by the target molecule are ameliorated. The dosage should notbe so large as to cause adverse side effects, such as, hyperviscositysyndromes, pulmonary edema, congestive heart failure, and other adverseside effects known in the art. Generally, the dosage will vary with theage, weight, height, body surface area, state of health (e.g., renal andliver function), condition, sex and extent of the disease in the patientand can routinely be determined by one of ordinary skill in the art. Thedosage can be adjusted by the individual physician in the event of anycomplication.

The dosage schedule and amounts effective for therapeutic andprophylactic uses, i.e., the “dosing regimen”, will depend upon avariety of factors, including the cause, stage and severity of thedisease or disorder, the health, physical status, age of the subjectbeing treated, and the site and mode of the delivery of the γPAMNcomposition. Therapeutic efficacy and toxicity of the γPAMN compositioncan be determined by standard pharmaceutical, pharmacological, andtoxicological procedures in cell cultures or experimental animals. Dataobtained from these procedures can likewise be used in formulating arange of dosages for human use. Moreover, therapeutic index (i.e., thedose therapeutically effective in 50 percent of the population dividedby the dose lethal to 50 percent of the population (ED50/LD50)) canreadily be determined using known procedures. The dosage is preferablywithin a range of concentrations that includes the ED50 with little orno toxicity, and may vary within this range depending on the dosage formemployed, sensitivity of the patient, and the route of administration.

The dosage regimen also takes into consideration pharmacokineticsparameters known in the art, such as, drug absorption rate,bioavailability, metabolism and clearance (see, e.g., Hidalgo-Aragones,J. Steroid Biochem. Mol. Biol. 58:611-617 (1996); Groning et al.,Pharmazie 51:337-341 (1996); Fotherby, Contraception 54:59-69 (1996);and Johnson et al., J. Pharm. Sci. 84:1144-1146 (1995)). It is wellwithin the state of the art for the clinician to determine the dosageregimen for each subject being treated. Moreover, single or multipleadministrations of the γPAMN composition can be administered dependingon the dosage and frequency as required and tolerated by the subject.The duration of prophylactic and therapeutic treatment will varydepending on the particular disease or condition being treated. Somediseases are amenable to acute treatment whereas others requirelong-term, chronic therapy. The γPAMN composition can be administeredserially, or simultaneously with the additional therapeutic agent.

In some embodiments, the γPAMN composition is administered in aliposomal composition at a dose of between 0.005 and 5000 mg of γPAMNper square meter of body surface area, or any range therein between. Infurther embodiments, the γPAMN composition is administered in aliposomal composition at a dose of between 0.1 and 1000 mg γPAMN persquare meter of body surface area, or any range therein between.

In some embodiments, the γPAMN composition is administered in animmunoconjugate composition at a dose of 1 mg/kg to 500 mg/kg, 1 mg/kgto 250 mg/kg, 1 mg/kg to 200 mg/kg, 1 mg/kg to 150 mg/kg, 1 mg/kg to 100mg/kg,′ mg/kg to 50 mg/kg, 1 mg/kg to 25 mg/kg, 1 mg/kg to 20 mg/kg, 1mg/kg to 15 mg/kg, 1 mg/kg to 10 mg/kg, or 1 mg/kg to 5 mg/kg, or anyrange therein between.

In another embodiment, the γPAMN composition is administered incombination with one or more additional therapeutics.

In some embodiment, the PLp-γPAMN and/or targeted-PLp-γPAMN is preparedas an infusion composition, an injection composition, a parenteralcomposition, or a topical composition. In further embodiments, theinjection includes one or more of: intraperitoneal injection, directintratumor injection, intra-arterial injection, and intravenousinjection, subcutaneous injection, intramuscular injection, delivery viatranscutaneous and intranasal route. In a further embodiment, thePLp-γPAMN and/or targeted-PLp-γPAMN is a liquid solution or asuspension. However, solid forms suitable for solution in, or suspensionin, liquid vehicles prior to injection are also provided herein. In someembodiments, the targeted pegylated liposomal gamma polyglutamatedaminopterin composition is formulated as an enteric-coated tablet or gelcapsule according to methods known in the art.

In some embodiments, the targeted pegylated liposomal gammapolyglutamated aminopterin formulations are administered to a tumor ofthe central nervous system using a slow, sustained intracranial infusionof the liposomes directly into the tumor (e.g., a convection-enhanceddelivery (CED)). See, Saito et al., Cancer Research 64:2572-2579 (2004);Mamot et al., J. Neuro-Oncology 68:1-9 (2004). In other embodiments, theformulations are directly applied to tissue surfaces. Sustained release,pH dependent release, and other specific chemical or environmentalcondition-mediated release administration of the pegylated liposomalgamma polyglutamated aminopterin formulations (e.g., depot injectionsand erodible implants) are also provided. Examples of suchrelease-mediating compositions are further described herein or otherwiseknown in the art.

For administration by inhalation, the compositions can be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,ichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount.

When it is desirable to deliver the compositions systemically, they canformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection can bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers. Pharmaceutical parenteral formulations include aqueoussolutions of the ingredients. Aqueous injection suspensions can containsubstances which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. Alternatively,suspensions of liposomes can be prepared as oil-based suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides.

Alternatively, the non-targeted or targeted pegylated liposomal gammapolyglutamated aminopterin can be in powder form or lyophilized form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The provided compositions (e.g., gamma polyglutamated aminopterin andliposomes containing the gamma polyglutamated aminopterin) can also beformulated in rectal or vaginal compositions such as suppositories orretention enemas, e.g., containing conventional suppository bases suchas cocoa butter or other glycerides.

Methods of Use and Treatment

In additional embodiments, the disclosure provides methods of usinggamma polyglutamated aminopterin (γPAMN) compositions. In someembodiments, the gamma γPAMN compositions are used to treat a disease ordisorder.

In some embodiments, the disclosure provides a method of killing a cellthat comprises contacting the cell with a composition comprising a gammapolyglutamated aminopterin (e.g., an γPAMN disclosed herein). In someembodiments, the contacted cell is a mammalian cell. In furtherembodiments, the contacted cell is a human cell. In some embodiments,the contacted cell is a hyperproliferative cell. In further embodiments,the hyperproliferative cell is a cancer cell. In yet furtherembodiments, the cancer cell is a primary cell or a cell from a cellline obtained/derived from a cancer selected from the group consistingof: a non-hematologic malignancy including such as for example, lungcancer, pancreatic cancer, breast cancer, ovarian cancer, prostatecancer, head and neck cancer, gastric cancer, gastrointestinal cancer,colorectal cancer, esophageal cancer, cervical cancer, liver cancer,kidney cancer, biliary duct cancer, gallbladder cancer, bladder cancer,sarcoma (e.g., osteosarcoma), brain cancer, central nervous systemcancer, and melanoma; and a hematologic malignancy such as for example,a leukemia, a lymphoma and other B cell malignancies, myeloma and otherplasma cell dysplasias or dyscrasias. In yet further embodiments, thecancer cell is a primary cell or a cell from a cell lineobtained/derived from a cancer selected from colorectal cancer, lungcancer, breast cancer, head and neck cancer, and pancreatic cancer.

In some embodiments, the method is performed in vivo. In otherembodiments, the method is performed in vitro. In some embodiments, theγPAMN composition contains 4, 5, 2-10, 4-6, or more than 5, γ-glutamylgroups. In some embodiments, the γPAMN composition comprises gammapentaglutamated aminopterin. In some embodiments, the γPAMN compositioncomprises gamma hexaglutamated aminopterin. In some embodiments, theγPAMN composition comprises L gamma polyglutamated aminopterin. In someembodiments, the γPAMN composition comprises D gamma polyglutamatedaminopterin. In some embodiments, the γPAMN composition comprises L andD gamma polyglutamated aminopterin.

In additional embodiments, the disclosure provides a method of killing acell that comprises contacting the cell with a liposome containing gammapolyglutamated aminopterin (e.g., an Lp-γPAMN such as, PLp-γPAMN,NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN or TPLp-γPAMN disclosed herein). Insome embodiments, the liposome is pegylated (e.g., PLp-γPAMN andNTPLp-γPAMN). In some embodiments, the liposome comprises a targetingmoiety on its surface that has specific affinity for an epitope of anantigen on the surface of the cell (e.g., TLp-γPAMN and TPLp-γPAMN). Infurther embodiments, the liposome is pegylated and comprises a targetingmoiety on its surface that specifically binds an antigen on the surfaceof the cell (e.g., TPLp-γPAMN). In some embodiments, the liposome is notpegylated (e.g., PLp-γPAMN and NTPLp-γPAMN). In some embodiments, theunpegylated liposome comprises a targeting moiety on its surface thatspecifically binds an antigen on the surface of the cell (e.g.,TLp-γPAMN and TPLp-γPAMN). In some embodiments, the contacted cell is amammalian cell. In further embodiments, the contacted cell is a humancell. In additional embodiments, the contacted cell is ahyperproliferative cell. In further embodiments, the hyperproliferativecell is a cancer cell. In further embodiments, the contacted cancer cellis a primary cell or a cell from a cell line obtained/derived from acancer selected from the group consisting of: lung cancer (e.g.,non-small cell), pancreatic cancer, breast cancer, ovarian cancer,prostate cancer, head and neck cancer, gastric cancer, gastrointestinalcancer, colorectal cancer, esophageal cancer, cervical cancer, livercancer, kidney cancer, biliary duct cancer, gallbladder cancer, bladdercancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervoussystem cancer, melanoma, myeloma, a leukemia and a lymphoma. In someembodiments, the method is performed in vivo. In other embodiments, themethod is performed in vitro. In some embodiments, the liposome containsa γPAMN containing 4, 5, 2-10, 4-6, or more than 5, γ-glutamyl groups.In some embodiments, liposome comprises L gamma polyglutamatedaminopterin. In some embodiments, the liposome comprises a γPAMNcontaining 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamylgroups in the L-form. In some embodiments, liposome comprises D gammapolyglutamated aminopterin. In some embodiments, the liposome comprisesa γPAMN containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10,γ-glutamyl groups in the D-form. In some embodiments, the liposomecomprises L and D gamma polyglutamated aminopterin. In some embodiments,the liposome comprises a γPAMN containing 2, 3, 4, 5, or more than 5,γ-glutamyl groups in the L-form, and 1, 2, 3, 4, 5 or more than 5,γ-glutamyl groups in the D-form. In some embodiments, the liposomecomprises gamma tetraglutamated aminopterin. In some embodiments, theliposome comprises gamma pentaglutamated aminopterin. In otherembodiments, the liposome comprises gamma hexaglutamated aminopterin.

In some embodiments, the disclosure provides a method of killing ahyperproliferative cell that comprises contacting a hyperproliferativecell with a delivery vehicle (e.g., a liposome or antibody) comprisinggamma polyglutamated aminopterin (e.g., an γPAMN disclosed herein). Insome embodiments, the delivery vehicle is an antibody (e.g., afull-length IgG antibody, a bispecific antibody, or a scFv). In someembodiments, the delivery vehicle is a liposome (e.g., an Lp-γPAMN suchas, PLp-γPAMN, NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN, or TPLp-γPAMN)). Insome embodiments, the delivery vehicle is non-targeted. In otherembodiments, the delivery vehicle is targeted and comprises a targetingmoiety on its surface that has specific affinity for an epitope on anantigen on the surface of the hyperproliferative cell. In furtherembodiments, the delivery vehicle comprises a targeting moiety that hasspecific affinity for an epitope on an antigen on the surface of thehyperproliferative cell selected from the group consisting of GONMB,TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin,Fibronectin Extra-domain B (ED-B), VEGFR2 (CD309), Tenascin, CollagenIV, Periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, the deliveryvehicle comprises a targeting moiety that has specific affinity for anepitope on a cell surface antigen(s) derived from, or determined to beexpressed on, a specific subject's cancer (tumor) such as a neoantigen.In some embodiments, the method is performed in vivo. In someembodiments, the method is performed in vitro. In some embodiments, thedelivery vehicle comprises an γPAMN containing 4, 5, 2-10, 4-6, or morethan 5, γ-glutamyl groups. In some embodiments, the delivery vehiclecomprises L gamma polyglutamated aminopterin. In some embodiments, thedelivery vehicle comprises a γPAMN containing 2, 3, 4, 5, 6, 7, 8, 9,10, or more than 10, γ-glutamyl groups in the L-form. In someembodiments, the delivery vehicle comprises D gamma polyglutamatedaminopterin. In some embodiments, the delivery vehicle comprises a γPAMNcontaining 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamylgroups in the D-form. In some embodiments, the delivery vehiclecomprises L and D gamma polyglutamated aminopterin. In some embodiments,the delivery vehicle comprises a γPAMN containing 2, 3, 4, 5, or morethan 5, γ-glutamyl groups in the L-form, and 1, 2, 3, 4, 5 or more than5, γ-glutamyl groups in the D-form. In some embodiments, the deliveryvehicle comprises gamma tetraglutamated aminopterin. In someembodiments, the delivery vehicle comprises gamma pentaglutamatedaminopterin. In other embodiments, the delivery vehicle comprises gammahexaglutamated aminopterin.

In particular embodiments, the method of a killing a hyperproliferativecell is performed using a liposome delivery vehicle that comprises gammapolyglutamated aminopterin (e.g., an Lp-γPAMN such as, PLp-γPAMN,NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN or TPLp-γPAMN disclosed herein). Insome embodiments, the delivery vehicle is an non-targeted liposome. Insome embodiments, the delivery vehicle comprises a targeting moiety onits surface that has specific affinity for an epitope on an antigen onthe surface of the hyperproliferative cell (e.g., TLp-γPAMN andTPLp-γPAMN). In some embodiments the delivery vehicle is a liposomecomprising a targeting moiety on its surface that has specific affinityfor an epitope on an antigen on the surface of the hyperproliferativecell (e.g., TLp-γPAMN and TPLp-γPAMN). In some embodiments, the deliveryvehicle is a liposome comprising a targeting moiety on its surface thathas specific affinity for an epitope on an antigen on the surface of thehyperproliferative cell. In further embodiments, the targeting moietyhas specific affinity for an epitope on an antigen selected from thegroup consisting of GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folatereceptor (e.g., folate receptor-γ, folate receptor-β or folatereceptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, Nectin 4,ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9(Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, Tissue factor, LIV-1(ZIP6), CGEN-15027, P Cadherin, Fibronectin Extra-domain B (ED-B),VEGFR2 (CD309), Tenascin, Collagen IV, Periostin, endothelin receptor,HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6,IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10,SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20,CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56,CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R,IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA1, EphA2, EphA3,EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, anintegrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242 antigen, Apo2,PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1,VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK,ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK. In someembodiments, the targeting moiety has specific affinity for an epitopeon a cell surface antigen(s) derived from, or determined to be expressedon, a specific subject's cancer (tumor) such as a neoantigen. In someembodiments, the liposome is pegylated (e.g., PLp-γPAMN, andNTPLp-γPAMN). In further embodiments, the liposome is pegylated andcomprises a targeting moiety on its surface that has specific affinityfor an epitope on an antigen on the surface of the hyperproliferativecell (e.g., TPLp-γPAMN). In other embodiments, the embodiments, theliposome is unpegylated. In some embodiments, the liposome isunpegylated and the liposome comprises a targeting moiety on its surfacethat has specific affinity for an epitope on an antigen on the surfaceof the hyperproliferative cell (e.g., TPLp-γPAMN). In some embodiments,the liposome comprises a γPAMN containing 4, 5, 2-10, 4-6, or more than5, γ-glutamyl groups. In some embodiments, liposome comprises L gammapolyglutamated aminopterin. In some embodiments, the liposome comprisesa γPAMN containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10,γ-glutamyl groups in the L-form. In some embodiments, liposome comprisesD gamma polyglutamated aminopterin. In some embodiments, the liposomecomprises a γPAMN containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than10, γ-glutamyl groups in the D-form. In some embodiments, the liposomecomprises L and D gamma polyglutamated aminopterin. In some embodiments,the liposome comprises an γPAMN containing 2, 3, 4, 5, or more than 5,γ-glutamyl groups in the L-form, and 1, 2, 3, 4, 5 or more than 5,γ-glutamyl groups in the D-form. In some embodiments, the liposomecomprises gamma tetraglutamated aminopterin. In some embodiments, theliposome comprises gamma pentaglutamated aminopterin. In otherembodiments, the liposome comprises gamma hexaglutamated aminopterin.

In additional embodiments, the disclosure provides a method ofinhibiting the proliferation of a cancer cell that comprises contactingthe cancer cell with a delivery vehicle (e.g., a liposome or antibody)comprising gamma polyglutamated aminopterin (e.g., an γPAMN disclosedherein). In some embodiments, the delivery vehicle is an antibody (e.g.,a full-length IgG antibody, a bispecific antibody, or a scFv). In someembodiments, the delivery vehicle is a liposome (e.g., an Lp-γPAMN suchas, PLp-γPAMN, NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN, or TPLp-γPAMN)). Insome embodiments, the delivery vehicle is non-targeted. In someembodiments, the delivery vehicle is targeted and comprises a targetingmoiety on its surface that has specific affinity for an epitope on anantigen on the surface of the cancer cell. In further embodiments, thedelivery vehicle comprises a targeting moiety that has specific affinityfor an epitope on a cell surface antigen selected from the groupconsisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor(e.g., folate receptor-α, folate receptor-β or folate receptor-δ), Mucin1 (MUC-1), MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclaseC (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, PCadherin, Fibronectin Extra-domain B (ED-B), VEGFR2 (CD309), Tenascin,Collagen IV, Periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR,EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3,FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6,CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30,CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, the deliveryvehicle comprises a targeting moiety that has specific affinity for anepitope on a cell surface antigen(s) derived from, or determined to beexpressed on, a specific subject's cancer (tumor) such as a neoantigen.In some embodiments, the delivery vehicle is an antibody that hasspecific affinity for an epitope on an antigen on the surface of thecancer cell. In some embodiments, the contacted cancer cell is amammalian cell. In further embodiments, the contacted cancer cell is ahuman cell. In additional embodiments, the contacted cancer cell is aprimary cell or a cell from a cell line obtained/derived from a cancerselected from the group consisting of: lung cancer (e.g., non-smallcell), pancreatic cancer, breast cancer, ovarian cancer, prostatecancer, head and neck cancer, gastric cancer, gastrointestinal cancer,colorectal cancer, esophageal cancer, cervical cancer, liver cancer,kidney cancer, biliary duct cancer, gallbladder cancer, bladder cancer,sarcoma (e.g., osteosarcoma), brain cancer, central nervous systemcancer, melanoma, myeloma, a leukemia and a lymphoma. In someembodiments, the method is performed in vivo. In some embodiments, themethod is performed in vitro. In some embodiments, the delivery vehicleis an antibody that has specific affinity for an epitope on one of theabove-listed cell surface antigens. In other embodiments, the targetingvehicle is a liposome that comprises a targeting moiety that hasspecific affinity for an epitope on the surface of the cancer cell. Inother embodiments, the targeting vehicle is a liposome that comprises atargeting moiety that has specific affinity for an epitope on one of theabove-listed cell surface antigens. In some embodiments, the deliveryvehicle is a liposome that is pegylated. In other embodiments, thedelivery vehicle is a liposome that is unpegylated. In some embodiments,the delivery vehicle comprises a γPAMN composition containing 4, 5,2-10, 4-6, or more than 5, γ-glutamyl groups. In some embodiments, thedelivery vehicle comprises L gamma polyglutamated aminopterin. In someembodiments, the delivery vehicle comprises a γPAMN containing 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10, γ-glutamyl groups in the L-form. Insome embodiments, the delivery vehicle comprises D gamma polyglutamatedaminopterin. In some embodiments, the delivery vehicle comprises a γPAMNcontaining 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamylgroups in the D-form. In some embodiments, the delivery vehiclecomprises L and D gamma polyglutamated aminopterin. In some embodiments,the delivery vehicle comprises an γPAMN containing 2, 3, 4, 5, or morethan 5, γ-glutamyl groups in the L-form, and 1, 2, 3, 4, 5 or more than5, γ-glutamyl groups in the D-form. In some embodiments, the deliveryvehicle comprises gamma tetraglutamated aminopterin. In someembodiments, the delivery vehicle comprises gamma pentaglutamatedaminopterin. In other embodiments, the delivery vehicle comprises gammahexaglutamated aminopterin.

In further embodiments, the disclosure provides a method of inhibitingthe proliferation of a cancer cell that comprises contacting the cancercell with a liposome comprising gamma polyglutamated aminopterin (e.g.,an γPAMN disclosed herein). In some embodiments, the liposome isnon-targeted. In some embodiments, the liposome is targeted andcomprises a targeting moiety on its surface that has specific affinityfor an epitope on an antigen on the surface of the cancer cell. Infurther embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope on a cell surface antigen selected fromthe group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, afolate receptor (e.g., folate receptor-α, folate receptor-β or folatereceptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, Nectin 4,ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9(Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, Tissue factor, LIV-1(ZIP6), CGEN-15027, P Cadherin, Fibronectin Extra-domain B (ED-B),VEGFR2 (CD309), Tenascin, Collagen IV, Periostin, endothelin receptor,HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6,IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10,SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20,CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56,CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R,IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA1, EphA2, EphA3,EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, anintegrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242 antigen, Apo2,PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1,VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK,ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK. In someembodiments, the liposome comprises a targeting moiety that has specificaffinity for an epitope on a cell surface antigen(s) derived from, ordetermined to be expressed on, a specific subject's cancer (tumor) suchas a neoantigen. In some embodiments, the contacted cancer cell is amammalian cell. In further embodiments, the contacted cancer cell is ahuman cell. In additional embodiments, the contacted cancer cell is aprimary cell or a cell from a cell line obtained/derived from a cancerselected from the group consisting of: lung cancer (e.g., non-smallcell), pancreatic cancer, breast cancer, ovarian cancer, prostatecancer, head and neck cancer, gastric cancer, gastrointestinal cancer,colorectal cancer, esophageal cancer, cervical cancer, liver cancer,kidney cancer, biliary duct cancer, gallbladder cancer, bladder cancer,sarcoma (e.g., osteosarcoma), brain cancer, central nervous systemcancer, melanoma, myeloma, a leukemia and a lymphoma. In someembodiments, the method is performed in vivo. In some embodiments, themethod is performed in vitro. In other embodiments, the targetingvehicle is a liposome that comprises a targeting moiety that hasspecific affinity for an epitope on one of the above-listed cell surfaceantigens. In some embodiments, the liposome is pegylated. In otherembodiments, the liposome that is unpegylated. In some embodiments, theliposome comprises an γPAMN composition containing 4, 5, 2-10, 4-6, ormore than 5, γ-glutamyl groups. In some embodiments, the liposomecomprises L gamma polyglutamated aminopterin. In some embodiments, theliposome comprises a γPAMN containing 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore than 10, γ-glutamyl groups in the L-form. In some embodiments, theliposome comprises D gamma polyglutamated aminopterin. In someembodiments, the liposome comprises a γPAMN containing 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more than 10, γ-glutamyl groups in the D-form. In someembodiments, the liposome comprises L and D gamma polyglutamatedaminopterin. In some embodiments, the liposome comprises an γPAMNcontaining 2, 3, 4, 5, or more than 5, γ-glutamyl groups in the L-form,and 1, 2, 3, 4, 5 or more than 5, γ-glutamyl groups in the D-form. Insome embodiments, the liposome comprises gamma tetraglutamatedaminopterin. In some embodiments, the liposome comprises gammapentaglutamated aminopterin. In other embodiments, the liposomecomprises gamma hexaglutamated aminopterin.

In additional embodiments, the disclosure provides a method for treatinga hyperproliferative disorder that comprises administering an effectiveamount of a delivery vehicle (e.g., antibody or liposome) comprisinggamma polyglutamated aminopterin (e.g., an γPAMN disclosed herein) to asubject having or at risk of having a hyperproliferative disorder. Insome embodiments, the delivery vehicle is an antibody (e.g., afull-length IgG antibody, a bispecific antibody, or a scFv). In someembodiments, the delivery vehicle is a liposome (e.g., an Lp-γPAMN suchas, PLp-γPAMN, NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN, or TPLp-γPAMN)). Insome embodiments, the administered delivery vehicle is pegylated. Insome embodiments, the administered delivery vehicle is not pegylated. Inadditional embodiments, the administered delivery vehicle comprises atargeting moiety that has specific affinity for an epitope of antigen onthe surface of the hyperproliferative cell. In additional embodiments,the delivery vehicle comprises a targeting moiety that has specificaffinity for an epitope of a cell surface antigen selected from thegroup consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folatereceptor (e.g., folate receptor-α, folate receptor-β or folatereceptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, Nectin 4,ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9(Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, Tissue factor, LIV-1(ZIP6), CGEN-15027, P Cadherin, Fibronectin Extra-domain B (ED-B),VEGFR2 (CD309), Tenascin, Collagen IV, Periostin, endothelin receptor,HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6,IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10,SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20,CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56,CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R,IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA1, EphA2, EphA3,EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, anintegrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242 antigen, Apo2,PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1,VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK,ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK. In someembodiments, the delivery vehicle comprises a targeting moiety that hasspecific affinity for an epitope on a cell surface antigen(s) derivedfrom, or determined to be expressed on, a specific subject's cancer(tumor) such as a neoantigen. In some embodiments, the targeting moietyis an antibody or an antigen binding antibody fragment. In someembodiments, the administered delivery vehicle does not comprise atargeting moiety that has a specific affinity for an epitope on a cellsurface antigen of the hyperproliferative cell. In some embodiments, theadministered delivery vehicle comprises γPAMN containing 4, 5, 2-10,4-6, or more than 5, γ-glutamyl groups. In some embodiments, theadministered delivery vehicle comprises L gamma polyglutamatedaminopterin. In some embodiments, the delivery vehicle comprises a γPAMNcontaining 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamylgroups in the L-form. In some embodiments, the delivery vehiclecomprises D gamma polyglutamated aminopterin. In some embodiments, thedelivery vehicle comprises a γPAMN containing 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more than 10, γ-glutamyl groups in the D-form. In someembodiments, the delivery vehicle comprises L and D gamma polyglutamatedaminopterin. In some embodiments, the delivery vehicle comprises anγPAMN containing 2, 3, 4, 5, or more than 5, γ-glutamyl groups in theL-form, and 1, 2, 3, 4, 5 or more than 5, γ-glutamyl groups in theD-form. In some embodiments, the administered delivery vehicle comprisesgamma tetraglutamated aminopterin. In some embodiments, the administereddelivery vehicle comprises gamma pentaglutamated aminopterin. In otherembodiments, the administered delivery vehicle comprises gammahexaglutamated aminopterin. In some embodiments, the hyperproliferativedisorder is cancer. In some embodiments, the hyperproliferative disorderis an autoimmune disease (e.g., inflammation and rheumatoid arthritis).In some embodiments, the hyperproliferative disorder is a benign ormalignant tumor; leukemia, hematological, or lymphoid malignancy. Inother embodiments, the hyperproliferative disorder selected from thegroup consisting of a: neuronal, glial, astrocytal, hypothalamic,glandular, macrophagal, epithelial, stromal, blastocoelic, inflammatory,angiogenic and immunologic disorder, including an autoimmune disease.

In additional embodiments, the disclosure provides a method for treatinga hyperproliferative disorder that comprises administering an effectiveamount of a liposome comprising gamma polyglutamated aminopterin (e.g.,an Lp-γPAMN such as, PLp-γPAMN, NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN, orTPLp-γPAMN) to a subject having or at risk of having ahyperproliferative disorder. In some embodiments, the liposome ispegylated. In some embodiments, the liposome is not pegylated. Inadditional embodiments, the liposome comprises a targeting moiety thathas a specific affinity for an epitope of an antigen on the surface ofthe hyperproliferative cell. In additional embodiments, the liposomecomprises a targeting moiety that has a specific affinity for an epitopeon a cell surface antigen selected from the group consisting of: GONMB,TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin,Fibronectin Extra-domain B (ED-B), VEGFR2 (CD309), Tenascin, CollagenIV, Periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, the liposomecomprises a targeting moiety that has a specific affinity for an epitopeon cell surface antigen(s) derived from, or determined to be expressedon, a specific subject's cancer (tumor) such as a neoantigen. In someembodiments, the targeting moiety is an antibody or an antigen bindingantibody fragment. In some embodiments, the liposome does not comprise atargeting moiety that has a specific affinity for an epitope on a cellsurface antigen of the hyperproliferative cell. In some embodiments, theliposome comprises γPAMN containing 4, 5, 2-10, 4-6, or more than 5,γ-glutamyl groups. In some embodiments, the liposome comprises L gammapolyglutamated aminopterin. In some embodiments, the liposome comprisesa γPAMN containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10,γ-glutamyl groups in the L-form. In some embodiments, liposome comprisesD gamma polyglutamated aminopterin. In some embodiments, the liposomecomprises a γPAMN containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than10, γ-glutamyl groups in the D-form. In some embodiments, the liposomecomprises L and D gamma polyglutamated aminopterin. In some embodiments,the liposome comprises an γPAMN containing 2, 3, 4, 5, or more than 5,γ-glutamyl groups in the L-form, and 1, 2, 3, 4, 5 or more than 5,γ-glutamyl groups in the D-form. In some embodiments, the liposomecomprises gamma tetraglutamated aminopterin. In some embodiments, theliposome comprises gamma pentaglutamated aminopterin. In otherembodiments, the liposome comprises gamma hexaglutamated aminopterin. Insome embodiments, the hyperproliferative disorder is cancer. In someembodiments, the hyperproliferative disorder is an autoimmune disease(e.g., rheumatoid arthritis). In some embodiments, thehyperproliferative disorder is a benign or malignant tumor; leukemia,hematological, or lymphoid malignancy. In other embodiments, thehyperproliferative disorder is selected from the group consisting of a:neuronal, glial, astrocytal, hypothalamic, glandular, macrophagal,epithelial, stromal, blastocoelic, inflammatory, angiogenic andimmunologic disorder, including an autoimmune disease.

Exemplary hyperproliferative disorders that can be treated according tothe disclosed methods include, but are not limited to, disordersassociated with benign, pre-malignant, and malignant cellularproliferation, including but not limited to, neoplasms and tumors (e.g.,histiocytoma, glioma, astrocytoma, osteoma), cancers (e.g., lung cancer,small cell lung cancer, gastrointestinal cancer, bowel cancer,colorectal cancer, breast carcinoma, ovarian carcinoma, prostate cancer,testicular cancer, liver cancer, kidney cancer, bladder cancer,pancreatic cancer, brain cancer, sarcoma (e.g., osteosarcoma, Kaposi'ssarcoma), and melanoma), leukemias, psoriasis, bone diseases,fibroproliferative disorders (e.g., of connective tissues), andatherosclerosis.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a deliveryvehicle (e.g., antibody or liposome) comprising gamma polyglutamatedaminopterin (e.g., an γPAMN disclosed herein) to a subject having or atrisk of having cancer. In some embodiments, the delivery vehicle is anantibody (e.g., a full-length IgG antibody, a bispecific antibody, or ascFv). In some embodiments, the delivery vehicle is a liposome (e.g., anLp-γPAMN such as, PLp-γPAMN, NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN, orTPLp-γPAMN). In some embodiments, the administered delivery vehicle ispegylated. In some embodiments, the administered delivery vehicle is notpegylated. In additional embodiments, the administered delivery vehiclecomprises a targeting moiety that has a specific affinity for an epitopeof antigen on the surface of a cancer cell. In additional embodiments,the delivery vehicle comprises a targeting moiety that has a specificaffinity for an epitope of a cell surface antigen selected from thegroup consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folatereceptor (e.g., folate receptor-α, folate receptor-β or folatereceptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, Nectin 4,ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9(Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, Tissue factor, LIV-1(ZIP6), CGEN-15027, P Cadherin, Fibronectin Extra-domain B (ED-B),VEGFR2 (CD309), Tenascin, Collagen IV, Periostin, endothelin receptor,HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6,IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10,SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20,CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56,CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R,IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA1, EphA2, EphA3,EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, anintegrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242 antigen, Apo2,PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1,VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK,ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK. In someembodiments, the delivery vehicle comprises a targeting moiety that hasspecific affinity for an epitope on a cell surface antigen(s) derivedfrom, or determined to be expressed on, a specific subject's cancer(tumor) such as a neoantigen. In some embodiments, the targeting moietyis an antibody or an antigen binding antibody fragment. In someembodiments, the administered delivery vehicle comprises γPAMNcontaining 4, 5, 2-10, 4-6, or more than 5, γ-glutamyl groups. In someembodiments, the administered delivery vehicle comprises L gammapolyglutamated aminopterin. In some embodiments, the administereddelivery vehicle comprises D gamma polyglutamated aminopterin. In someembodiments, the administered delivery vehicle comprises L and D gammapolyglutamated aminopterin. In some embodiments, the administereddelivery vehicle comprises gamma tetraglutamated aminopterin. In someembodiments, the administered delivery vehicle comprises gammapentaglutamated aminopterin. In other embodiments, the administereddelivery vehicle comprises gamma hexaglutamated aminopterin. In someembodiments, the cancer is selected from the group consisting of: lung(e.g., non-small lung cancer), pancreatic, breast cancer, ovarian, lung,prostate, head and neck, gastric, gastrointestinal, colon, esophageal,cervical, kidney, biliary duct, gallbladder, and a hematologicmalignancy (e.g., a leukemia or lymphoma).

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomecomprising gamma polyglutamated aminopterin (e.g., an Lp-γPAMN such as,PLp-γPAMN, NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN, or TPLp-γPAMN) to asubject having or at risk of having cancer. In some embodiments, theliposome is pegylated. In some embodiments, the liposome is notpegylated. In additional embodiments, the liposome comprises a targetingmoiety that has a specific affinity for an epitope of antigen on thesurface of a cancer cell. In additional embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeof a cell surface antigen selected from the group consisting of: GONMB,TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin,Fibronectin Extra-domain B (ED-B), VEGFR2 (CD309), Tenascin, CollagenIV, Periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeon a cell surface antigen(s) derived from, or determined to be expressedon, a specific subject's cancer (tumor) such as a neoantigen. In someembodiments, the targeting moiety is an antibody or an antigen bindingantibody fragment. In some embodiments, the liposome comprises γPAMNcontaining 4, 5, 2-10, 4-6, or more than 5, γ-glutamyl groups. In someembodiments, the liposome comprises L gamma polyglutamated aminopterin.In some embodiments, the liposome comprises a γPAMN containing 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10, γ-glutamyl groups in the L-form. Insome embodiments, the liposome comprises D gamma polyglutamatedaminopterin. In some embodiments, the liposome comprises a γPAMNcontaining 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamylgroups in the D-form. In some embodiments, the liposome comprises L andD gamma polyglutamated aminopterin. In some embodiments, the liposomecomprises an γPAMN containing 2, 3, 4, 5, or more than 5, γ-glutamylgroups in the L-form, and 1, 2, 3, 4, 5 or more than 5, γ-glutamylgroups in the D-form. In some embodiments, the liposome comprises gammatetraglutamated aminopterin. In some embodiments, the liposome comprisesgamma pentaglutamated aminopterin. In other embodiments, the liposomecomprises gamma hexaglutamated aminopterin. In some embodiments, thecancer is selected from the group consisting of: lung (e.g., non-smalllung cancer), pancreatic, breast cancer, ovarian, lung, prostate, headand neck, gastric, gastrointestinal, colon, esophageal, cervical,kidney, biliary duct, gallbladder, and a hematologic malignancy (e.g., aleukemia or lymphoma).

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering to a subject having or at risk ofhaving cancer, an effective amount of a liposomal composition containinga liposome that comprises gamma polyglutamated aminopterin and atargeting moiety that has a specific affinity for an epitope of antigenon the surface of the cancer. In some embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeof a cell surface antigen selected from the group consisting of: GONMB,TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin,Fibronectin Extra-domain B (ED-B), VEGFR2 (CD309), Tenascin, CollagenIV, Periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeon a cell surface antigen(s) derived from, or determined to be expressedon, a specific subject's cancer (tumor) such as a neoantigen. In someembodiments, the administered liposomal composition comprises pegylatedliposomes (e.g., TPLp-γPAMN). In some embodiments, the administeredliposomal composition comprises liposomes that are not pegylated. Insome embodiments, liposomes of the administered liposomal compositioncomprises γPAMN containing 4, 5, 2-10, 4-6, or more than 5, γ-glutamylgroups. In some embodiments, a liposome of the liposomal compositioncomprises L gamma polyglutamated aminopterin. In some embodiments, aliposome of the liposomal composition comprises D gamma polyglutamatedaminopterin. In some embodiments, a liposome of the liposomalcomposition comprises L and D gamma polyglutamated aminopterin. In someembodiments, a liposome of the liposomal composition comprises a γPAMNcontaining 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamylgroups in the L-form. In some embodiments, a liposome of the liposomalcomposition comprises D gamma polyglutamated aminopterin. In someembodiments, a liposome of the liposomal composition comprises a γPAMNcontaining 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamylgroups in the D-form. In some embodiments, the liposome comprises L andD gamma polyglutamated aminopterin. In some embodiments a liposome ofthe liposomal composition comprises γPAMN containing 2, 3, 4, 5, or morethan 5, γ-glutamyl groups in the L-form, and 1, 2, 3, 4, 5 or more than5, γ-glutamyl groups in the D-form. In some embodiments, liposomes ofthe administered liposomal composition comprise gamma tetraglutamatedaminopterin. In some embodiments, liposomes of the administeredliposomal composition comprise gamma pentaglutamated aminopterin. Inother embodiments, liposomes of the administered liposomal compositioncomprises gamma hexaglutamated aminopterin. In some embodiments, theliposomal composition is administered to treat a cancer selected fromthe group consisting of: lung cancer, pancreatic, breast cancer, ovariancancer, lung cancer, prostate cancer, head and neck cancer, gastriccancer, gastrointestinal cancer, colon cancer, esophageal cancer,cervical cancer, kidney cancer, biliary duct cancer, gallbladder cancer,and a hematologic malignancy.

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of a tumor specific antigen (TSA) ortumor associated antigen (TAA). In some embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeof an antigen selected from the group consisting of: a tumordifferentiation antigen (e.g., MART1/MelanA, gp100 (Pmel 17),tyrosinase, TRP1, and TRP2), a tumor-specific multilineage antigen(e.g., MAGE1, MAGE3, BAGE, GAGE1, GAGE2, and p15), an overexpressedembryonic antigen (e.g., carcinoembryonic antigen (CEA)), anoverexpressed oncogene or mutated tumor-suppressor gene product (e.g.,p53, Ras, and HER2/neu), a unique tumor antigen resulting fromchromosomal translocations (e.g., BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, andMYL-RAR), a viral antigen (e.g., Epstein Barr virus antigen EBVA, humanpapillomavirus (HPV) antigen E6 or E7), GP 100), prostatic acidphosphatase (PAP), prostate-specific antigen (PSA), PTGER4, ITGA4, CD37,CD52, CD62L (L-selectin), CXCR4, CD69, EVI2B (CD361), SLC39A8, MICB,LRRC70, CLELC2B, HMHA1, LST1, and CMTM6 (CKLFSF6).

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of a hematologic tumor antigen. Infurther embodiments, the targeting moiety has specific affinity for anepitope of a hematologic tumor antigen selected from the groupconsisting of: CD19, CD20, CD22, CD30, CD138, CD33, CD38, CD123, CS1,ROR1, Lewis^(Y), Ig kappa light chain, TCR, BCMA, TACI, BAFFR (CD268),CALLA, and a NKG2DL ligand). In some embodiments, the liposome comprisesa targeting moiety that has specific affinity for an epitope of a B-celllymphoma-specific idiotype immunoglobulin, or a B-cell differentiationantigen (e.g., CD19, CD20, and CD37). In some embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeof an antigen on a multiple myeloma cell (e.g., CS-1, CD38, CD138, MUC1,HM1.24, CYP1B1, SP17, PRAME, Wilms' tumor 1 (WT1), and heat shockprotein gp96) or an antigen on myeloid cells (e.g., TSLPR and IL-7R).

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of a solid tumor antigen. In furtherembodiments, the targeting moiety has specific affinity for an epitopeof a hematologic tumor antigen selected from the group consisting of:disialoganglioside (GD2), o-acetyl GD2, EGFRvIII, ErbB2, VEGFR2, FAP,mesothelin, IL13Ra2 (glioma), cMET, PSMA, L1CAM, CEA, and EGFR.

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of an antigen selected from the groupconsisting of: CD137, PDL1, CTLA4, CD47, KIR, TNFRSF10B (DR5), TIM3,PD1, cMet, Glycolipid F77, EGFRvIII, HLAA2 (NY-ESO-1), LAG3, CD134(OX40), HVEM, BTLA, TNFRSF25 (DR3), CD133, MAGE A3, PSCA, MUC1, CD44v6,CD44v6/7, CD44v7/8, IL11Ra, ephA2, CAIX, MNCAIX, CSPG4, MUC16, EPCAM(EGP2), TAG72, EGP40, ErbB receptor family, ErbB2 (HER2), ErbB3/4,RAGE1, GD3, FAR, Lewis^(Y), NCAM, HLAA1/MAGE1, MAGEA1, MAGEA3, MAGE-A4,B7H3, WT1, MelanA (MART1), HPV E6, HPV E7, thyroglobulin, tyrosinase,PSA, CLL1GD3, Tn Ag, FLT3, KIT, PRSS21, CD24, PDGFR-beta, SSEA4,prostase, PAP, ELF2M, ephB2, IGF1, IGFII, IGFI receptor, LMP2, gp100,bcr-abl, Fucosyl GM1, sLe, GM3, TGS5, folate receptor beta, TEM1(CD248), TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD7a, HLE, CD179a,ALK, Plysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3,GPR20, LY6K, OR51E2, TARP, LAGEla, legumain, E7, ETV6-AML, sperm protein17, XAGE1, Tie 2, MAD-CT1, MAD-CT2, Fos-related antigen 1, p53, p53mutant, prostein, survivin, telomerase, PCTA1 (Galectin 8), Ras mutant,hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETSfusion gene), NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC,TRP2, CYP1B1, BORIS, SART3, PAXS, OY-TES1, LCK, AKAP4, SSX2, reversetranscriptase, RU1, RU2, intestinal carboxyl esterase, neutrophilelastase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF,CLEC12A, BST2, EMR2, LY75, GPC3, FCRLS, IGLL1, TSP-180, MAGE4, MAGE5,MAGE6, VEGFR1, IGF1R, hepatocyte growth factor receptor, p185ErbB2,p180ErbB-3, nm-23H1, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,beta-Catenin, CDK4, Mum1, p15, p16, 43-9F, 5T4, 791Tgp72, β-humanchorionic gonadotropin, BCA225, BTAA, CA125, CA15-3, CA 27.29 (BCAA),CA195, CA242, CA-50, CAM43, CD68, CO-029, FGF5, G250, HTgp-175, M344,MA50, MG7-Ag, MOV18, NB/70K, NY-CO1, RCAS1, SDCCAG16, M2BP, TAAL6, TLP,and TPS, glioma-associated antigen, gamma-fetoprotein (AFP), p26fragment of AFP, lectin-reactive AFP, and TLR4.

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of an antigen selected from the groupconsisting of: PDGFRA, VEGFR1, VEGFR3, neuropilin 1 (NRP1), neuropilin 2(NRP2), betacellulin, PLGF, RET (rearranged during transfection), TIE1,TIE2 (TEK), CA125, CD3, CD4, CD7, CD10, CD13, CD25 CD32, CD32b, CD44(e.g., CD44v6), CD47, CD49e (integrin gamma 5), CD54 (ICAM), CD55, CD64,CD74, CD80, CD90, CD200, CD147, CD166, CD200, ESA, SHH, DHH, IHH,patched 1 (PTCH1), smoothened (SMO), WNT1, WNT2B, WNT3A, WNT4, WNT4A,WNT5A, WNT5B, WNT7B, WNT8A, WNT10A, WNT10B, WNT16B, LKP5, LRP5, LRP6,FZD1, FZD2, FZD4, FZD5, FZD6, FZD7, FZD8, Notch, Notch1, Notch3, Notch4,DLL4, Jagged, Jagged1, Jagged2, Jagged3, TNFRSF1A (TNFR1, p55, p60),TNFRSF1B (TNFR2), TNFRSF6 (Fas, CD95), TNFRSF6B (DcR3), TNFRSF7 (CD27),TNFSF9 (41BB Ligand), TNFRSF8 (CD30), TNFRSF10A (TRAILR1, DR4),TNFRSF11A (RANK), TNFRSF12 (TWEAKR), TNFRSF19L (KELT), TNFRSF19 (TROY),TNFRSF21 (DR6), ILIRI, 1L1R₂, IL2R, IL5R, IL6R, 1L8R, IL10R, IL12R,IL13R, IL15R, IL18R, IL19R, IL21R, IL23R, XAG1, XAG3, REGIV, FGFR1,FGFR2, FGFR3, ALK, ALK1, ALK7, ALCAM, Axl, TGFb, TGFb2, TGFb3, TGFBR1,IGFIIR, BMPRI, N-cadherin, E-cadherin, VE-cadherin, ganglioside GM2,ganglioside GD3, PSGR, DCC, CDCP1, CXCR2, CXCR7, CCR3, CCR4, CCR5, CCR7,CCR10, Claudin1, Claudin2, Claudin3, Claudin4, TMEFF2, neuregulin, MCSF,CSF, CSFR (fms), GCSF, GCSFR, BCAM, BRCA1, BRCA2, HLA-DR, ABCC3, ABCB5,HM 1.24, LFA1, LYNX, S100A8, S100A9, SCF, Von Willebrand factor, LewisY6 receptor, CA G250 (CA9), CRYPTO, VLA5, HLADR, MUC18, mucin CanAg,EGFL7, integrin avb3, integrin γ5β activin B1 gamma, leukotriene B4receptor (LTB4R), neurotensin NT receptor (NTR), 5T4 oncofetal antigen,Tenascin C, MMP, MMP2, MMP7, MMP9, MMP12, MMP14, MMP26, cathepsin G,SULF1, SULF2, MET, CA9, TM4SF1, syndecan (SDCl), Ephrin B4, TEM1,TGFbeta 1, and TGFBRII.

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of an antigen associated with adisorder of the immune system (e.g., an autoimmune disorder and aninflammatory disorder), or is associated with regulating an immuneresponse. In some embodiments, the targeting moiety has specificaffinity for an epitope of a cell surface antigen expressed on thesurface of a macrophage (expressing CD44).

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of an immunoinhibitory target. Inanother embodiment, the AD is an epitope of an immunoinhibitory targetselected from the group consisting of: IL1Ra, IL6R, CD26L, CD28, CD80,FcGamma RIIB. In another embodiment, the AD in the Adapter is an epitopeof an immunostimulatory target selected from: CD25, CD28, CTLA4, PD1,B7H1 (PDL1), B7H4 TGFbeta, TNFRSF4 (OX40), TNFRSF5 (CD40), TNFRSF9(41BB, CD137), TNFRSF14 (HVEM), TNFRSF25 (DR3), and TNFRSF18 (GITR).

In some embodiments, the liposome comprises a targeting moiety that hasspecific affinity for an epitope of an antigen selected from the groupconsisting of: IL1Rb, C3AR, C5AR, CXCR1, CXCR2, CCR1, CCR3, CCR7, CCR8,CCR9, CCR10, ChemR23, MPL, GP130, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8,TLR9, TREM1, TREM2, CD49a (integrin gamma 1), integrin a5b3, gamma4integrin subunit, A4B7 integrin, cathepsin G, TNFRSF3 (LTBR), TNFRSF6(Fas, CD95), TNFRSF6B (DcR3), TNFRSF8 (CD30), TNFRSF11A (RANK), TNFRSF16(NGFR), TNFRSF19L (RELT), TNFRSF19 (TROY), TNFRSF21 (DR6), CD14, CD23,CD36, CD36L, CD39, CD91, CD153, CD164, CD200, CD200R, B71 (CD80), B72(CD86), B7h, B7DC (PDL2), ICOS, ICOSL, MHC, CD, B7H2, B7H3, B7x, SLAM,KIM1, SLAMF2, SLAMF3, SLAMF4, SLAMF5, SLAMF6, SLAMF7, TNFRSF1A (TNFR1,p55, p60), TNFRSF1B (TNFR2), TNFRSF7 (CD27), TNFRSF12 (TWEAKR), TNFRSF5(CD40), IL1R, IL2R, IL4Ra, IL5R, IL6RIL15R, IL17R, IL17Rb, IL17RC,IL22RA, IL23R, TSLPR, B7RP1, cKit, GMCSF, GMCSFR, CD2, CD4, CD11a, CD18,CD30, CD40, CD86, CXCR3, CCR2, CCR4, CCR5, CCR8, RhD, IgE, and Rh.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomalcomposition to a subject having or at risk of having a cancer thatexpresses folate receptor on its cell surface, wherein the liposomalcomposition comprises liposomes that comprise (a) gamma polyglutamatedaminopterin (γPAMN) and (b) a targeting moiety that has specific bindingaffinity for a folate receptor. In some embodiments, the targetingmoiety has specific binding affinity for folate receptor alpha (FR-α),folate receptor beta (FR-β), and/or folate receptor delta (FR-δ). Insome embodiments, the targeting moiety has a specific binding affinityfor folate receptor alpha (FR-α), folate receptor beta (FR-β), and/orfolate receptor delta (FR-δ). In some embodiments, the targeting moietyhas a specific binding affinity for folate receptor alpha (FR-α) andfolate receptor beta (FR-β). In some embodiments, the administeredliposomal composition comprises pegylated liposomes (e.g., TPLp-γPAMN).In some embodiments, the administered liposomal composition comprisesliposomes that are not pegylated. In some embodiments, liposomes of theadministered liposomal composition comprises γPAMN containing 4, 5,2-10, 4-6, or more than 5, γ-glutamyl groups. In some embodiments, aliposome of the liposomal composition comprises L gamma polyglutamatedaminopterin. In some embodiments, a liposome of the liposomalcomposition comprises a γPAMN containing 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore than 10, γ-glutamyl groups in the L-form. In some embodiments, aliposome of the liposomal composition comprises D gamma polyglutamatedaminopterin. In some embodiments, a liposome of the liposomalcomposition comprises a γPAMN containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more than 10, γ-glutamyl groups in the D-form. In some embodiments, aliposome of the liposomal composition comprises L and D gammapolyglutamated aminopterin. In some embodiments, a liposome of theliposomal composition comprises a γPAMN containing 2, 3, 4, 5, or morethan 5, γ-glutamyl groups in the L-form, and 1, 2, 3, 4, 5 or more than5, γ-glutamyl groups in the D-form. In some embodiments, a liposome ofthe liposomal composition comprises tetraglutamated aminopterin. In someembodiments, a liposome of the liposomal composition comprisespentaglutamated aminopterin. In some embodiments, a liposome of theliposomal composition comprises hexaglutamated aminopterin.

In some embodiments, a liposome of the liposomal composition comprisesan γPAMN containing 2, 3, 4, 5, or more than 5, γ-glutamyl groups. Insome embodiments, liposomes of the administered liposomal compositioncomprise gamma tetraglutamated aminopterin. In some embodiments,liposomes of the administered liposomal composition comprise gammapentaglutamated aminopterin. In some embodiments, liposomes of theadministered liposomal composition comprises gamma hexaglutamatedaminopterin. In some embodiments, the liposomal composition isadministered to treat an epithelial tissue malignancy. In someembodiments, the liposomal composition is administered to treat a cancerselected from the group consisting of: lung cancer, pancreatic, breastcancer, ovarian cancer, lung cancer, prostate cancer, head and neckcancer, gastric cancer, gastrointestinal cancer, colon cancer,esophageal cancer, cervical cancer, kidney cancer, biliary duct cancer,gallbladder cancer, and a hematologic malignancy.

In some embodiments, the disclosure provides a method for treating lungcancer (e.g., non-small lung cancer) that comprises administering aneffective amount of a delivery vehicle (e.g., an antibody or liposome)comprising gamma polyglutamated aminopterin (e.g., an γPAMN disclosedherein) to a subject having or at risk of having lung cancer. Inparticular embodiments, the, the cancer is non-small cell lung cancer.In some embodiments, the delivery vehicle is an antibody (e.g., afull-length IgG antibody, a bispecific antibody, or a scFv). In someembodiments, the delivery vehicle is a liposome (e.g., an Lp-γPAMN suchas, PLp-γPAMN, NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN, or TPLp-γPAMN)). Insome embodiments, the administered delivery vehicle is pegylated. Insome embodiments, the administered delivery vehicle is not pegylated. Inadditional embodiments, the delivery vehicle comprises a targetingmoiety on its surface that has specific affinity for an epitope on anantigen on the surface of a lung cancer (e.g., non-small cell lungcancer) cell. In further embodiments, the delivery vehicle comprises atargeting moiety that has specific affinity for an epitope on an antigenselected from the group consisting of Mucin 1, Nectin 4, NaPi2b, CD56,EGFR, and SC-16. In some embodiments, the targeting moiety is anantibody or a fragment of an antibody. In additional embodiments, thedelivery vehicle is a liposome, and the liposome comprises a targetingmoiety that has specific affinity for an epitope on an antigen selectedfrom the group consisting of Mucin 1, Nectin 4, NaPi2b, CD56, EGFR, andSC-16. In further embodiments, the delivery vehicle is a pegylatedliposome that comprises a targeting moiety that has specific affinityfor an epitope on an antigen selected from consisting of Mucin 1, Nectin4, NaPi2b, CD56, EGFR, and SC-16. In some embodiments, the administereddelivery vehicle comprises γPAMN containing 4, 5, 2-10, 4-6, or morethan 5, glutamyl groups. In some embodiments, the administered deliveryvehicle comprises L gamma polyglutamated aminopterin. In someembodiments, the administered delivery vehicle comprises a γPAMNcontaining 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamylgroups in the L-form. In some embodiments, the delivery vehiclecomprises D gamma polyglutamated aminopterin. In some embodiments, theadministered delivery vehicle comprises D gamma polyglutamatedaminopterin. In some embodiments, the delivery vehicle comprises a γPAMNcontaining 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamylgroups in the D-form. In some embodiments, the delivery vehiclecomprises L and D gamma polyglutamated aminopterin. In some embodiments,the delivery vehicle comprises an γPAMN containing 2, 3, 4, 5, or morethan 5, γ-glutamyl groups in the L-form, and 1, 2, 3, 4, 5 or more than5, γ-glutamyl groups in the D-form. In some embodiments, theadministered delivery vehicle comprises gamma tetraglutamatedaminopterin. In some embodiments, the administered delivery vehiclecomprises gamma pentaglutamated aminopterin. In other embodiments, theadministered delivery vehicle comprises gamma hexaglutamatedaminopterin.

In some embodiments, the disclosure provides a method for treatingpancreatic cancer that comprises administering an effective amount of adelivery vehicle (e.g., an antibody (ADC) or liposome) comprising gammapolyglutamated aminopterin (e.g., an γPAMN disclosed herein) to asubject having or at risk of having pancreatic cancer. In someembodiments, the delivery vehicle is an antibody (e.g., a full-lengthIgG antibody, a bispecific antibody, or a scFv). In some embodiments,the delivery vehicle is a liposome (e.g., an Lp-γPAMN such as,PLp-γPAMN, NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN, or TPLp-γPAMN)). In someembodiments, the administered delivery vehicle is pegylated. In someembodiments, the administered delivery vehicle is not pegylated. Inadditional embodiments, the delivery vehicle comprises a targetingmoiety on its surface that has specific affinity for an epitope on anantigen on the surface of a pancreatic cancer cell. In furtherembodiments, the delivery vehicle comprises a targeting moiety that hasspecific affinity for an epitope on an antigen selected from the groupconsisting of TACSTD2 (TROP2), Mucin 1, mesothelin, Guanylyl cyclase C(GCC), SLC44A4, and Nectin 4. In further embodiments, the deliveryvehicle is a liposome, and the liposome comprises a targeting moiety hasspecific affinity for an epitope on an antigen selected from the groupconsisting of TACSTD2 (TROP2), Mucin 1, Mesothelin, Guanylyl cyclase C(GCC), SLC44A4, and Nectin 4. In some embodiments, the administereddelivery vehicle comprises γPAMN containing 4, 5, 2-10, 4-6, or morethan 5, γ-glutamyl groups. In some embodiments, the administereddelivery vehicle comprises L gamma polyglutamated aminopterin. In someembodiments, the delivery vehicle comprises a γPAMN containing 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10, γ-glutamyl groups in the L-form. Insome embodiments, the delivery vehicle comprises D gamma polyglutamatedaminopterin. In some embodiments, the delivery vehicle comprises a γPAMNcontaining 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamylgroups in the D-form. In some embodiments, the delivery vehiclecomprises L and D gamma polyglutamated aminopterin. In some embodiments,the delivery vehicle comprises an γPAMN containing 2, 3, 4, 5, or morethan 5, γ-glutamyl groups in the L-form, and 1, 2, 3, 4, 5 or more than5, γ-glutamyl groups in the D-form. In some embodiments, theadministered delivery vehicle comprises gamma tetraglutamatedaminopterin. n some embodiments, the administered delivery vehiclecomprises gamma pentaglutamated aminopterin. In other embodiments, theadministered delivery vehicle comprises gamma hexaglutamatedaminopterin.

In additional embodiments, the disclosure provides a method for treatingbreast cancer (e.g., triple negative breast cancer (estrogen receptor⁻,progesterone receptor⁻, and HER2)) that comprises administering aneffective amount of a delivery vehicle (e.g., an antibody or liposome)comprising gamma polyglutamated aminopterin (e.g., an γPAMN disclosedherein) to a subject having or at risk of having breast cancer. In someembodiments, the administered delivery vehicle is a liposome thatcomprises gamma polyglutamated aminopterin. In some embodiments, thedelivery vehicle is an antibody (e.g., a full-length IgG antibody, abispecific antibody, or a scFv). In some embodiments, the deliveryvehicle is a liposome (e.g., an Lp-γPAMN such as, PLp-γPAMN, NTLp-γPAMN,NTPLp-γPAMN, TLp-γPAMN, or TPLp-γPAMN)). In some embodiments, theadministered delivery vehicle is pegylated. In some embodiments, theadministered delivery vehicle is not pegylated. In additionalembodiments, the delivery vehicle comprises a targeting moiety on itssurface that has specific affinity for an epitope on an antigen on thesurface of a breast cancer cell. In further embodiments, the deliveryvehicle comprises a targeting moiety that has specific affinity for anepitope on an antigen selected from the group consisting of: LIV-1(ZIP6), EGFR, HER2, HER3, Mucin 1, GONMB, and Nectin 4. In someembodiments, the targeting moiety is an antibody or a fragment of anantibody. In additional embodiments, the delivery vehicle is a liposome,and the liposome comprises a targeting moiety that has specific affinityfor an epitope on an antigen selected from the group consisting of:LIV-1 (ZIP6), EGFR, HER2, HER3, Mucin 1, GONMB, and Nectin 4. In someembodiments, the administered delivery vehicle comprises γPAMNcontaining 4, 5, 2-10, 4-6, or more than 5, γ-glutamyl groups. In someembodiments, the delivery vehicle comprises L gamma polyglutamatedaminopterin. In some embodiments, the delivery vehicle comprises a γPAMNcontaining 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamylgroups in the L-form. In some embodiments, the delivery vehiclecomprises D gamma polyglutamated aminopterin. In some embodiments, thedelivery vehicle comprises a γPAMN containing 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more than 10, γ-glutamyl groups in the D-form. In someembodiments, the delivery vehicle comprises L and D gamma polyglutamatedaminopterin. In some embodiments, the delivery vehicle comprises anγPAMN containing 2, 3, 4, 5, or more than 5, γ-glutamyl groups in theL-form, and 1, 2, 3, 4, 5 or more than 5, γ-glutamyl groups in theD-form. In some embodiments, the administered delivery vehicle comprisesgamma tetraglutamated aminopterin. In some embodiments, the administereddelivery vehicle comprises gamma pentaglutamated aminopterin. In someembodiments, the administered delivery vehicle comprises gammahexaglutamated aminopterin.

In some embodiments, the disclosure provides a method for treating ahematological cancer that comprises administering an effective amount ofa delivery vehicle (e.g., an antibody or liposome) comprising gammapolyglutamated aminopterin (e.g., an γPAMN disclosed herein) to asubject having or at risk of having a hematological cancer. In someembodiments, the delivery vehicle is an antibody (e.g., a full-lengthIgG antibody, a bispecific antibody, or a scFv). In some embodiments,the delivery vehicle is a liposome (e.g., an Lp-γPAMN such as,PLp-γPAMN, NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN, or TPLp-γPAMN)). In someembodiments, the administered delivery vehicle is pegylated. In someembodiments, the administered delivery vehicle is not pegylated. Inadditional embodiments, the delivery vehicle comprises a targetingmoiety on its surface that has specific affinity for an epitope on anantigen on the surface of a hematological cancer cell. In furtherembodiments, the delivery vehicle comprises a targeting moiety that hasspecific affinity for an epitope on an antigen selected from the groupconsisting of: CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33,and CD98. In further embodiments, the delivery vehicle is a liposome,and the liposome comprises a targeting moiety has specific affinity foran epitope on an antigen selected from the group consisting of: CD30,CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, and CD98. In someembodiments, the administered delivery vehicle comprises γPAMNcontaining 4, 5, 2-10, 4-6, or more than 5, γ-glutamyl groups. In someembodiments, the delivery vehicle comprises L gamma polyglutamatedaminopterin. In some embodiments, the delivery vehicle comprises a γPAMNcontaining 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamylgroups in the L-form. In some embodiments, the delivery vehiclecomprises D gamma polyglutamated aminopterin. In some embodiments, thedelivery vehicle comprises a γPAMN containing 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more than 10, γ-glutamyl groups in the D-form. In someembodiments, the delivery vehicle comprises L and D gamma polyglutamatedaminopterin. In some embodiments, the delivery vehicle comprises anγPAMN containing 2, 3, 4, 5, or more than 5, γ-glutamyl groups in theL-form, and 1, 2, 3, 4, 5 or more than 5, γ-glutamyl groups in theD-form. In some embodiments, the administered delivery vehicle comprisesgamma tetraglutamated aminopterin. In some embodiments, the administereddelivery vehicle comprises gamma pentaglutamated aminopterin. In otherembodiments, the administered delivery vehicle comprises gammahexaglutamated aminopterin.

In some embodiments, the disclosure provides a method for treating asubject having or at risk of having a cancer that is distinguishable bythe expression of an antigen on its cell surface. Thus, in someembodiments, the disclosure provides a method for treating cancer thatcomprises administering to a subject having or at risk of having acancer, an effective amount of a delivery vehicle (e.g., an antibody orliposome) comprising a targeting moiety that has specific affinity foran epitope on a surface antigen of the cancer and gamma polyglutamatedaminopterin (e.g., an γPAMN disclosed herein). In some embodiments, theadministered delivery vehicle is pegylated. In some embodiments, thetargeting moiety is an antibody or a fragment of an antibody. Inadditional embodiments, the delivery vehicle is a liposome. In someembodiments, the administered delivery vehicle comprises γPAMNcontaining 4, 5, 2-10, 4-6, or more than 5, γ-glutamyl groups. In someembodiments, the delivery vehicle comprises L gamma polyglutamatedaminopterin. In some embodiments, the delivery vehicle comprises a γPAMNcontaining 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamylgroups in the L-form. In some embodiments, the delivery vehiclecomprises D gamma polyglutamated aminopterin. In some embodiments, thedelivery vehicle comprises a γPAMN containing 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more than 10, γ-glutamyl groups in the D-form. In someembodiments, the delivery vehicle comprises L and D gamma polyglutamatedaminopterin. In some embodiments, the delivery vehicle comprises anγPAMN containing 2, 3, 4, 5, or more than 5, γ-glutamyl groups in theL-form, and 1, 2, 3, 4, 5 or more than 5, γ-glutamyl groups in theD-form. In some embodiments, the administered delivery vehicle comprisesgamma tetraglutamated aminopterin. In some embodiments, the administereddelivery vehicle comprises gamma pentaglutamated aminopterin. In otherembodiments, the administered delivery vehicle comprises gammahexaglutamated aminopterin.

In some embodiments, the disclosed compositions (e.g., liposomescontaining gamma polyglutamated aminopterin) are administered tosubjects having or at risk of having a cancer, a solid tumor, and/or ametastasis that is distinguishable by the expression of a tumor specificantigen or tumor associated antigen on its cell surface. Thus, in someembodiments, the disclosure provides a method for treating cancer thatcomprises administering an effective amount of a delivery vehicle (e.g.,liposome) comprising a targeting moiety and gamma polyglutamatedaminopterin (e.g., an γPAMN disclosed herein) to a subject having or atrisk of having a cancer, solid tumor, and/or metastasis that isdistinguishable by the expression of a tumor specific antigen or tumorassociated antigen on its cell surface cancer, and wherein the targetingmoiety has specific binding affinity for an epitope on an tumor specificantigen or tumor associated antigen. In some embodiments, theadministered delivery vehicle is a liposome. In further embodiments, theliposome is pegylated. In additional embodiments, the delivery vehiclecomprises a targeting moiety that has specific affinity for an epitopeon a cell surface antigen expressed on the surface of a cancer, a solidtumor, and/or a metastatic cell. In additional embodiments, thetargeting moiety has specific affinity for an epitope on a cell surfaceantigen selected from the group consisting of: GONMB, TACSTD2 (TROP2),CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-α, folatereceptor-β or folate receptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1,mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b,CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16,Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin, FibronectinExtra-domain B (ED-B), VEGFR2 (CD309), Tenascin, Collagen IV, Periostin,endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2,FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7,FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15,CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38,CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138,cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA1,EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3,EphB4, EphB6, an integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA,c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB,TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the delivery vehicle comprises a targeting moietythat has specific affinity for an epitope on a cell surface antigen(s)derived from, or determined to be expressed on, a specific subject'scancer (tumor) such as a neoantigen. In some embodiments, theadministered delivery vehicle comprises γPAMN containing 4, 5, 2-10,4-6, or more than 5, γ-glutamyl groups. In some embodiments, thedelivery vehicle comprises a γPAMN containing 2, 3, 4, 5, 6, 7, 8, 9,10, or more than 10, γ-glutamyl groups in the L-form. In someembodiments, the delivery vehicle comprises a γPAMN containing 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamyl groups in the L-form.In some embodiments, the delivery vehicle comprises D gammapolyglutamated aminopterin. In some embodiments, the delivery vehiclecomprises a γPAMN containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than10, γ-glutamyl groups in the D-form. In some embodiments, the deliveryvehicle comprises L and D gamma polyglutamated aminopterin. In someembodiments, the delivery vehicle comprises an γPAMN containing 2, 3, 4,5, or more than 5, γ-glutamyl groups in the L-form, and 1, 2, 3, 4, 5 ormore than 5, γ-glutamyl groups in the D-form. In some embodiments, theadministered delivery vehicle comprises gamma tetraglutamatedaminopterin. In some embodiments, the administered delivery vehiclecomprises gamma pentaglutamated aminopterin. In other embodiments, theadministered delivery vehicle comprises gamma hexaglutamatedaminopterin.

In further embodiments, the delivery vehicle is a liposome, and theliposome comprises a targeting moiety that has specific affinity for acell surface antigen selected from the group consisting of: GONMB,TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin,Fibronectin Extra-domain B (ED-B), VEGFR2 (CD309), Tenascin, CollagenIV, Periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeon a cell surface antigen(s) derived from, or determined to be expressedon, a specific subject's cancer (tumor) such as a neoantigen. In someembodiments, the administered delivery vehicle comprises γPAMNcontaining 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In someembodiments, the administered delivery vehicle comprises gammapentaglutamated aminopterin. In other embodiments, the administereddelivery vehicle comprises gamma hexaglutamated aminopterin. In someembodiments, the administered delivery vehicle comprises L gammapolyglutamated aminopterin. In some embodiments, the administereddelivery vehicle comprises D gamma polyglutamated aminopterin. In someembodiments, the administered delivery vehicle comprises L and D gammapolyglutamated aminopterin.

In further embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a deliveryvehicle (e.g., an antibody or liposome) comprising a targeting moiety onits surface has specific affinity for an epitope of a folate receptor,and a gamma polyglutamated aminopterin (e.g., an γPAMN disclosed herein)to a subject having or at risk of having a cancer that contains cellsexpressing the folate receptor on their cell surface. In someembodiments, the targeting moiety is an antibody, or an antigen bindingfragment of an antibody. In further embodiments, the targeting moietyhas specific affinity for folate receptor alpha, folate receptor beta orfolate receptor delta. As disclosed herein, the folate receptor targetedpegylated liposomes containing gamma polyglutamated aminopterin are ableto deliver high quantities of gamma polyglutamated aminopterin to cancercells and particularly cancer cells that express folate receptors,compared to normal cells (i.e., cells that unlike cancer cells do notactively take up liposomes, and/or do not express folate receptors). Anycancers that express folate receptors may be treated according to thedisclosed methods. It should be noted that some cancers may expressfolate receptors in an early stage while the majority of cancers mayexpress folate receptors at late stages. In some embodiments, theadministered delivery vehicle is a liposome. In further embodiments, theliposome is pegylated. In some embodiments, the administered deliveryvehicle comprises γPAMN containing 4, 5, 2-10, 4-6, or more than 5,

In some embodiments, the delivery vehicle comprises L gammapolyglutamated aminopterin. In some embodiments, the delivery vehiclecomprises a γPAMN containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than10, γ-glutamyl groups in the L-form. In some embodiments, the deliveryvehicle comprises D gamma polyglutamated aminopterin. In someembodiments, the delivery vehicle comprises a γPAMN containing 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamyl groups in the D-form.In some embodiments, the delivery vehicle comprises L and D gammapolyglutamated aminopterin. In some embodiments, the delivery vehiclecomprises an γPAMN containing 2, 3, 4, 5, or more than 5, γ-glutamylgroups in the L-form, and 1, 2, 3, 4, 5 or more than 5, γ-glutamylgroups in the D-form. In some embodiments, the administered deliveryvehicle comprises gamma tetraglutamated aminopterin. In someembodiments, the administered delivery vehicle comprises gammapentaglutamated aminopterin. In other embodiments, the administereddelivery vehicle comprises gamma hexaglutamated aminopterin.

In additional embodiments, the disclosure provides a method for cancermaintenance therapy that comprises administering an effective amount ofa liposomal composition comprising liposomes that contain gammapolyglutamated aminopterin (e.g., an γPAMN disclosed herein) to asubject that is undergoing or has undergone cancer therapy. In someembodiments, the administered liposomal composition is a PLp-γPAMN,NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN or TPLp-γPAMN. In some embodiments,the administered liposomal composition comprises pegylated liposomes(e.g., PLp-γPAMN, NTPLp-γPAMN, or TPLp-γPAMN). In some embodiments, theadministered liposomal composition comprises a targeting moiety that hasspecific affinity for an epitope on a surface antigen of a cancer cell(e.g., TLp-γPAMN or TPLp-γPAMN). In some embodiments, the administeredliposomal composition comprises liposomes that are pegylated andcomprise a targeting moiety (e.g., TPLp-γPAMN). In some embodiments, theadministered liposomal composition comprises liposomes that comprise atargeting moiety and further comprises liposomes that do not comprise atargeting moiety. In some embodiments, the administered liposomalcomposition comprises liposomes that are pegylated and liposomes thatare not pegylated. In some embodiments, liposomes of the administeredliposomal composition comprise gamma polyglutamated aminopterin thatcontains 4, 5, 2-10, 4-6, or more than 5, γ-glutamyl groups. In someembodiments, the delivery vehicle comprises L and D gamma polyglutamatedaminopterin. In some embodiments, the delivery vehicle comprises a γPAMNcontaining 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamylgroups in the L-form. In some embodiments, the delivery vehiclecomprises D gamma polyglutamated aminopterin. In some embodiments, thedelivery vehicle comprises a γPAMN containing 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more than 10, γ-glutamyl groups in the D-form. In someembodiments, the delivery vehicle comprises L and D gamma polyglutamatedaminopterin. In some embodiments, the delivery vehicle comprises anγPAMN containing 2, 3, 4, 5, or more than 5, γ-glutamyl groups in theL-form, and 1, 2, 3, 4, 5 or more than 5, γ-glutamyl groups in theD-form. In some embodiments, a liposome of the administered liposomalcomposition comprises gamma tetraglutamated aminopterin. In someembodiments, a liposome of the administered liposomal compositioncomprises gamma pentaglutamated aminopterin. In other embodiments, aliposome of the administered liposomal composition comprises gammahexaglutamated aminopterin.

In some embodiments, the cancer treated by one or more of the methodsdisclosed herein is a solid tumor lymphoma. Examples of solid tumorlymphoma include Hodgkin's lymphoma, Non-Hodgkin's lymphoma, and B celllymphoma.

In some embodiments, the cancer treated by one or more of the methodsdisclosed herein is bone cancer, brain cancer, breast cancer, colorectalcancer, connective tissue cancer, cancer of the digestive system,endometrial cancer, esophageal cancer, eye cancer, cancer of the headand neck, gastric cancer, intra-epithelial neoplasm, melanomaneuroblastoma, Non-Hodgkin's lymphoma, non-small cell lung cancer,prostate cancer, retinoblastoma, or rhabdomyosarcoma.

In some embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a compositioncomprising a delivery vehicle and gamma polyglutamated aminopterin to asubject having or at risk of having cancer. In some embodiments, theadministered composition comprises a pegylated delivery vehicle. In someembodiments, the administered composition comprises a targeting moietythat has a specific affinity for an epitope of antigen on the surface ofa target cell of interest such as a cancer cell. In some embodiments,the delivery vehicle comprises an antibody or an antigen bindingantibody fragment. In some embodiments, the composition is administeredto treat a cancer selected from the group consisting of: lung cancer,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, headand neck cancer, gastric cancer, gastrointestinal cancer, colorectalcancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer,biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, melanoma,myeloma, a leukemia and a lymphoma. In some embodiments, theadministered composition contains 4, 5, 2-10, 4-6, or more than 5,γ-glutamyl groups. In some embodiments, the delivery vehicle comprises Lgamma polyglutamated aminopterin. In some embodiments, the deliveryvehicle comprises a γPAMN containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or morethan 10, γ-glutamyl groups in the L-form. In some embodiments, thedelivery vehicle comprises D gamma polyglutamated aminopterin. In someembodiments, the delivery vehicle comprises a γPAMN containing 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamyl groups in the D-form.In some embodiments, the delivery vehicle comprises L and D gammapolyglutamated aminopterin. In some embodiments, the delivery vehiclecomprises an γPAMN containing 2, 3, 4, 5, or more than 5, γ-glutamylgroups in the L-form, and 1, 2, 3, 4, 5 or more than 5, γ-glutamylgroups in the D-form. In some embodiments, the administered compositioncomprises gamma tetraglutamated aminopterin. In some embodiments, theadministered composition comprises gamma pentaglutamated aminopterin. Inother embodiments, the administered composition comprises gammahexaglutamated aminopterin

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomalcomposition comprising liposomes that contain gamma polyglutamatedaminopterin (e.g., Lp-γPAMN, PLp-γPAMN, NTLp-γPAMN, NTPLp-γPAMN,TLp-γPAMN or TPLp-γPAMN) to a subject having or at risk of havingcancer. In some embodiments, the liposomal composition is administeredto treat a cancer selected from the group consisting of: lung cancer,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, headand neck cancer, gastric cancer, gastrointestinal cancer, colorectalcancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer,biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, melanoma,myeloma, a leukemia and a lymphoma. In some embodiments, theadministered liposomal composition comprises pegylated liposomes (e.g.,PLp-γPAMN, NTPLp-γPAMN, or TPLp-γPAMN). In some embodiments, a liposomeof the administered liposomal composition comprises an γPAMN containing4, 5, 2-10, 4-6, or more than 5, γ-glutamyl groups. In some embodiments,a liposome of the liposomal composition comprises L gamma polyglutamatedaminopterin. In some embodiments, a liposome of the administeredliposomal composition comprises a γPAMN containing 2, 3, 4, 5, 6, 7, 8,9, 10, or more than 10, γ-glutamyl groups in the L-form. In someembodiments, a liposome of the liposomal composition comprises D gammapolyglutamated aminopterin. In some embodiments, a liposome of theliposomal composition comprises a γPAMN containing 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more than 10, γ-glutamyl groups in the D-form. In someembodiments, a liposome of the liposomal composition comprises L and Dgamma polyglutamated aminopterin. In some embodiments, a liposome of theliposomal composition comprises an γPAMN containing 2, 3, 4, 5, or morethan 5, γ-glutamyl groups in the L-form, and 1, 2, 3, 4, 5 or more than5, γ-glutamyl groups in the D-form. In some embodiments, a liposome ofthe administered liposomal composition comprise gamma tetraglutamatedaminopterin. In some embodiments, a liposome of the administeredliposomal composition comprise gamma pentaglutamated aminopterin. Inother embodiments, a liposome of the administered liposomal compositioncomprises gamma hexaglutamated aminopterin.

In additional embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomalcomposition that comprises targeted liposomes (e.g., TLp-γPAMN orTPLp-γPAMN) to a subject having or at risk of having cancer, wherein theliposomal composition comprises liposomes that comprise gammapolyglutamated aminopterin (Lp-γPAMN) and further comprise a targetingmoiety having a specific affinity for a surface antigen (epitope) on thecancer. In some embodiments, the liposomal composition is administeredto treat a cancer selected from the group consisting of: lung cancer,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, headand neck cancer, gastric cancer, gastrointestinal cancer, colorectalcancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer,biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g.,osteosarcoma), brain cancer, central nervous system cancer, melanoma,myeloma, a leukemia and a lymphoma. In some embodiments, theadministered liposomal composition comprises pegylated liposomes (e.g.,TPLp-γPAMN). In some embodiments, a liposome of the liposomalcomposition comprises L gamma polyglutamated aminopterin. In someembodiments, liposomes of the administered liposomal compositioncomprise an γPAMN containing 4, 5, 2-10, 4-6, or more than 5, γ-glutamylgroups. In some embodiments, a liposome of the administered liposomalcomposition comprises a γPAMN containing 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore than 10, γ-glutamyl groups in the L-form. In some embodiments, aliposome of the liposomal composition comprises D gamma polyglutamatedaminopterin. In some embodiments, a liposome of the liposomalcomposition comprises a γPAMN containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more than 10, γ-glutamyl groups in the D-form. In some embodiments, aliposome of the liposomal composition comprises L and D gammapolyglutamated aminopterin. In some embodiments, a liposome of theliposomal composition comprises an γPAMN containing 2, 3, 4, 5, or morethan 5, γ-glutamyl groups in the L-form, and 1, 2, 3, 4, 5 or more than5, γ-glutamyl groups in the D-form. In some embodiments, a liposome ofthe administered liposomal composition comprises gamma tetraglutamatedaminopterin. In some embodiments, a liposome of the administeredliposomal composition comprises gamma pentaglutamated aminopterin. Inother embodiments, a liposome of the administered liposomal compositioncomprises gamma hexaglutamated aminopterin.

In further embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of a liposomalcomposition that contains targeted liposomes (e.g., TLp-γPAMN orTPLp-γPAMN) to a subject having or at risk of having a cancer thatexpresses folate receptor on its cell surface, wherein the liposomalcomposition comprises liposomes that comprise (a) gamma polyglutamatedaminopterin (γPAMN) and (b) a targeting moiety that has specific bindingaffinity for the folate receptor. In some embodiments, the administeredliposomal composition comprises pegylated liposomes (e.g., TPLp-γPAMN).In some embodiments, the targeting moiety has a specific bindingaffinity for folate receptor alpha (FR-α), folate receptor beta (FR-β),and/or folate receptor delta (FR-δ). In some embodiments, the targetingmoiety has a specific binding affinity for folate receptor alpha (FR-α),folate receptor beta (FR-β), and/or folate receptor delta (FR-δ). Insome embodiments, the targeting moiety has a specific binding affinityfor folate receptor gamma (FR-late receptor beta (FR-β), and/or folatereceptor delta (FR-δ). In some embodiments, the targeting moiety has aspecific binding affinity for folate receptor alpha (FR-α) and folatereceptor beta (FR-β). In some embodiments, the liposomal composition isadministered to treat a cancer selected from the group consisting of:lung cancer, pancreatic, breast cancer, ovarian cancer, lung cancer,prostate cancer, head and neck cancer, gastric cancer, gastrointestinalcancer, colon cancer, esophageal cancer, cervical cancer, kidney cancer,biliary duct cancer, gallbladder cancer, and a hematologic malignancy Insome embodiments, a liposome of the administered liposomal compositioncomprise an γPAMN containing 4, 5, 2-10, 4-6, or more than 5, γ-glutamylgroups. In some embodiments, a liposome of the liposomal compositioncomprises L gamma polyglutamated aminopterin. In some embodiments, aliposome of the administered liposomal composition comprises a γPAMNcontaining 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamylgroups in the L-form. In some embodiments, a liposome of the liposomalcomposition comprises D gamma polyglutamated aminopterin. In someembodiments, a liposome of the liposomal composition comprises a γPAMNcontaining 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamylgroups in the D-form. In some embodiments, a liposome of the liposomalcomposition comprises L and D gamma polyglutamated aminopterin. In someembodiments, a liposome of the liposomal composition comprises an γPAMNcontaining 2, 3, 4, 5, or more than 5, γ-glutamyl groups in the L-form,and 1, 2, 3, 4, 5 or more than 5, γ-glutamyl groups in the D-form. Insome embodiments, a liposome of the administered liposomal compositioncomprises gamma tetraglutamated aminopterin. In some embodiments, aliposome of the administered liposomal composition comprises gammapentaglutamated aminopterin. In other embodiments, a liposome of theadministered liposomal compositions comprises gamma hexaglutamatedaminopterin.

In some embodiments, the disclosure provides a method for treating adisorder of the immune system (e.g., an autoimmune disease such asinflammation and rheumatoid arthritis) that comprises administering aneffective amount of a delivery vehicle (e.g., antibody or liposome)comprising gamma polyglutamated aminopterin (e.g., an γPAMN disclosedherein) to a subject having or at risk of having a disorder of theimmune system. In some embodiments, the delivery vehicle is an antibody(e.g., a full-length IgG antibody, a bispecific antibody, or a scFv). Insome embodiments, the delivery vehicle is a liposome (e.g., an Lp-γPAMNsuch as, PLp-γPAMN, NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN, or TPLp-γPAMN).In some embodiments, the administered delivery vehicle is pegylated. Insome embodiments, the administered delivery vehicle is not pegylated. Inadditional embodiments, the administered delivery vehicle comprises atargeting moiety that has a specific affinity for an epitope of antigenon the surface of an immune cell associated with a disorder of theimmune system. In some embodiments, the targeting moiety is an antibodyor an antigen binding antibody fragment. In some embodiments, theadministered delivery vehicle comprises γPAMN containing 4, 5, 2-10,4-6, or more than 5, γ-glutamyl groups. In some embodiments, thedelivery vehicle comprises L gamma polyglutamated aminopterin. In someembodiments, the delivery vehicle comprises a γPAMN containing 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10, γ-glutamyl groups in the L-form. Insome embodiments, the delivery vehicle comprises D gamma polyglutamatedaminopterin. In some embodiments, the delivery vehicle comprises a γPAMNcontaining 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, γ-glutamylgroups in the D-form. In some embodiments, the delivery vehiclecomprises L and D gamma polyglutamated aminopterin. In some embodiments,the delivery vehicle comprises an γPAMN containing 2, 3, 4, 5, or morethan 5, γ-glutamyl groups in the L-form, and 1, 2, 3, 4, 5 or more than5, γ-glutamyl groups in the D-form. In some embodiments, theadministered delivery vehicle comprises gamma tetraglutamatedaminopterin. In some embodiments, the administered delivery vehiclecomprises gamma pentaglutamated aminopterin. In other embodiments, theadministered delivery vehicle comprises gamma hexaglutamatedaminopterin. In some embodiments, the autoimmune disease is inflammationand rheumatoid arthritis.

In some embodiments, the disclosure provides a method for treating aninfectious disease (e.g., HIV) that comprises administering an effectiveamount of a delivery vehicle (e.g., antibody or liposome) comprisinggamma polyglutamated aminopterin (e.g., an γPAMN disclosed herein) to asubject having or at risk of having an infectious disease. In someembodiments, the delivery vehicle is an antibody (e.g., a full-lengthIgG antibody, a bispecific antibody, or a scFv). In some embodiments,the delivery vehicle is a liposome (e.g., an Lp-γPAMN such as,PLp-γPAMN, NTLp-γPAMN, NTPLp-γPAMN, TLp-γPAMN, or TPLp-γPAMN). In someembodiments, the administered delivery vehicle is pegylated. In someembodiments, the administered delivery vehicle is not pegylated. Inadditional embodiments, the administered delivery vehicle comprises atargeting moiety that has a specific affinity for an epitope of antigenon the surface of a pathogen associated with an infectious disease. Insome embodiments, the targeting moiety is an antibody or an antigenbinding antibody fragment. In some embodiments, the administereddelivery vehicle comprises γPAMN containing 4, 5, 2-10, 4-6, or morethan 5, γ-glutamyl groups. In some embodiments, the administereddelivery vehicle comprises L gamma polyglutamated aminopterin. In someembodiments, the delivery vehicle comprises a γPAMN containing 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10, γ-glutamyl groups in the L-form. Insome embodiments, the administered delivery vehicle comprises D gammapolyglutamated aminopterin. In some embodiments, the delivery vehiclecomprises a γPAMN containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than10, γ-glutamyl groups in the D-form. In some embodiments, theadministered delivery vehicle comprises L and D gamma polyglutamatedaminopterin. In some embodiments, the delivery vehicle comprises anγPAMN containing 2, 3, 4, 5, or more than 5, γ-glutamyl groups in theL-form, and 1, 2, 3, 4, 5 or more than 5, γ-glutamyl groups in theD-form. In some embodiments, the administered delivery vehicle comprisesgamma tetrapentaglutamated aminopterin. In some embodiments, theadministered delivery vehicle comprises gamma pentaglutamatedaminopterin. In other embodiments, the administered delivery vehiclecomprises gamma hexaglutamated aminopterin.

In some embodiments, the administered delivery vehicle is a liposome. Infurther embodiments, the liposome is pegylated. In additionalembodiments, the delivery vehicle comprises a targeting moiety on itssurface that specifically binds an antigen on the surface of a targetcell of interest. In further embodiments, the delivery vehicle comprisesa targeting moiety that has specific affinity for an epitope of a cellsurface antigen selected from the group consisting of: GONMB, TACSTD2(TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-α,folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1), MUC-6, STEAP1,mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC), SLC44A4, NaPi2b,CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16,Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin, FibronectinExtra-domain B (ED-B), VEGFR2 (CD309), Tenascin, Collagen IV, Periostin,endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2,FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7,FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15,CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38,CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138,cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, an EphB receptor, EphA1,EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3,EphB4, EphB6, an integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), a C242antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA,c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha., PDGFR beta, TrkA, TrkB,TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the delivery vehicle comprises a targeting moietythat has specific affinity for an epitope on a cell surface antigen(s)derived from, or determined to be expressed on, a specific subject'scancer (tumor) such as a neoantigen.

In further embodiments, the delivery vehicle is a liposome, and theliposome comprises a targeting moiety that has specific affinity for anepitope of a cell surface antigen selected from the group consisting of:GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folatereceptor-α, folate receptor-β or folate receptor-δ), Mucin 1 (MUC-1),MUC-6, STEAP1, mesothelin, Nectin 4, ENPP3, Guanylyl cyclase C (GCC),SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (Carbonic anhydrase), 5T4 (TPBG),SLTRK6, SC-16, Tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin,Fibronectin Extra-domain B (ED-B), VEGFR2 (CD309), Tenascin, CollagenIV, Periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII,FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11,CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33,CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98,CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 an EphA receptor, anEphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8,EphB1, EphB2, EphB3, EphB4, EphB6, an integrin (e.g., integrin αvβ3,αvβ5, or αvβ6), a C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2,endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFRalpha., PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7,Ryk, TCR, NMDAR, LNGFR, and MuSK. In some embodiments, the liposomecomprises a targeting moiety that has specific affinity for an epitopeon a cell surface antigen(s) derived from, or determined to be expressedon, a specific subject's cancer (tumor) such as a neoantigen.

In some embodiments, the disclosure provides for the use of acomposition comprising a gamma polyglutamated aminopterin formanufacture of a medicament for treatment of a hyperproliferativedisease. In some embodiments, the gamma polyglutamated aminopterincomprise 5 or more glutamyl groups. In some embodiments, the gammapolyglutamated aminopterin is pentaglutamated or hexaglutamated. In someembodiments, the gamma polyglutamated aminopterin is polyglutamatedaminopterin (AMN), aminopterin (AMN). In some embodiments, the gammapolyglutamated aminopterin is in a liposome. In some embodiments, thehyperproliferative disease is cancer. In some embodiments, the cancer isselected from the group consisting of: lung (e.g., non-small lungcancer), pancreatic, breast cancer, ovarian, lung, prostate, head andneck, gastric, gastrointestinal, colon, esophageal, cervical, kidney,biliary duct, gallbladder, and a hematologic malignancy. In someembodiments, the cancer is pancreatic cancer. In some embodiments, thecancer is breast cancer. In some embodiments, the cancer is pancreaticcancer. In some embodiments, the cancer is triple negative breastcancer. In some embodiments, the cancer is lung cancer. In someembodiments, the cancer is non-small cell lung cancer. In someembodiments, the cancer is leukemia or lymphoma. In some embodiments,the hyperproliferative disease is an autoimmune disease. In someembodiments, the hyperproliferative disease is inflammation andrheumatoid arthritis.

The disclosed methods can practiced in any subject that is likely tobenefit from delivery of compositions contemplated herein (e.g., gammapolyglutamated aminopterin compositions such as liposome containing agamma pentaglutamated or gamma hexaglutamated AMN). Mammalian subjects,and in particular, human subjects are preferred. In some embodiments,the subjects also include animals such as household pets (e.g., dogs,cats, rabbits, and ferrets), livestock or farm animals (e.g., cows,pigs, sheep, chickens and other poultry), horses such as thoroughbredhorses, laboratory animals (e.g., mice, rats, and rabbits), and othermammals. In other embodiments, the subjects include fish and otheraquatic species.

The subjects to whom the agents are delivered may be normal subjects.Alternatively the subject may have or be at risk of developing acondition that can be diagnosed or that can benefit from delivery of oneor more of the provided compositions. In some embodiments, suchconditions include cancer (e.g., solid tumor cancers or non-solid cancersuch as leukemias). In some embodiments, these conditions (e.g.,cancers) involve cells that express an antigen that can be specificallybound by a targeted pegylated liposomal gamma polyglutamated aminopterindisclosed herein. In further embodiments, these antigens specificallybind and internalize the targeted pegylated liposomal gammapolyglutamated aminopterin into the cell. In some embodiments, thetargeted pegylated liposomal gamma polyglutamated aminopterinspecifically binds a folate receptor (e.g., folate receptor alpha(FR-α), folate receptor beta (FR-β) and folate receptor delta (FR-δ))expressed on the surface of the cancer cell.

Tests for diagnosing the conditions that can be treated with theprovided compositions are known in the art and will be familiar to themedical practitioner. The determination of whether a cell type expressesfolate receptors can be made using commercially available antibodies.These laboratory tests include without limitation microscopic analyses,cultivation dependent tests (such as cultures), and nucleic aciddetection tests. These include wet mounts, stain-enhanced microscopy,immune microscopy (e.g., FISH), hybridization microscopy, particleagglutination, enzyme-linked immunosorbent assays, urine screeningtests, DNA probe hybridization, and serologic tests. The medicalpractitioner will generally also take a full history and conduct acomplete physical examination in addition to running the laboratorytests listed above.

A subject having a cancer can, for example, be a subject that hasdetectable cancer cells. A subject at risk of developing a cancer can,for example, be a subject that has a higher than normal probability ofdeveloping cancer. These subjects include, for instance, subjects havinga genetic abnormality that has been demonstrated to be associated with ahigher likelihood of developing a cancer, subjects having a familialdisposition to cancer, subjects exposed to cancer causing agents (e.g.,carcinogens) such as tobacco, asbestos, or other chemical toxins, andsubjects previously treated for cancer and in apparent remission.

In some embodiments, the disclosure provides methods for selectivelydeliver a folate receptor targeted pegylated liposomal gammapolyglutamated aminopterin to a tumor cell expressing a folate receptoron its surface at a rate that is higher (e.g., at least two-foldgreater, at least three-fold greater, at least four-fold greater, or atleast five-fold greater, than a cell not expressing folate receptor onits cell surface). In some embodiments, the delivered pegylated liposomecomprises gamma polyglutamated AMN. In some embodiments, the deliveredpegylated liposome comprises L-gamma polyglutamated AMN. In someembodiments, the delivered pegylated liposome comprises D-gammapolyglutamated AMN.

i. Combination Therapy

In certain embodiments, in addition to administering gammapolyglutamated AMN composition described herein, the method or treatmentfurther comprises administering at least one additional therapeuticagent. An additional therapeutic agent can be administered prior to,concurrently with, and/or subsequently to, administration of the gammapolyglutamated AMN composition. The additional therapeutic agent can beassociated with a gamma polyglutamated AMN delivery vehicle (e.g.,coencapsulated with gamma polyglutamated AMN in a liposome), present ina solution containing a gamma polyglutamated AMN delivery vehicle, or ina separate formulation from the composition containing the gammapolyglutamated AMN composition. Pharmaceutical compositions comprising apolypeptide or agent and the additional therapeutic agent(s) are alsoprovided. In some embodiments, the at least one additional therapeuticagent comprises 1, 2, 3, or more additional therapeutic agents.

Combination therapy with two or more therapeutic agents often usesagents that work by different mechanisms of action, although this is notrequired. Combination therapy using agents with different mechanisms ofaction may result in additive or synergetic effects. Combination therapymay allow for a lower dose of each agent than is used in monotherapy,thereby reducing toxic side effects and/or increasing the therapeuticindex of the polypeptide or agent(s). Combination therapy may decreasethe likelihood that resistant cancer cells will develop. In someembodiments, combination therapy comprises a therapeutic agent thataffects the immune response (e.g., enhances or activates the response)and a therapeutic agent that affects (e.g., inhibits or kills) thetumor/cancer cells.

In some embodiments, the disclosure provides a method for treatingcancer that comprises administering an effective amount of an gammapolyglutamated aminopterin composition disclosed herein and a biologic.In some embodiments, the gamma polyglutamated aminopterin isadministered in combination with a therapeutic antibody. In furtherembodiments, the gamma polyglutamated aminopterin is administered incombination with an anti-CD antibody (e.g., rituximab) or an antibodythat binds an immune checkpoint protein (e.g., CTLA4, PD1, PDL1, andTIM3). In further embodiments, the gamma polyglutamated aminopterin isadministered in combination with an fc-fusion protein (e.g.,entanercept).

In some embodiments, the disclosure provides a method for treatingdisorder of the immune system that comprises administering an effectiveamount of an gamma polyglutamated aminopterin composition disclosedherein and a biologic. In some embodiments, the gamma polyglutamatedaminopterin is administered in combination with a therapeutic antibody.In further embodiments, the gamma polyglutamated aminopterin isadministered in combination with an anti-TNF antibody (e.g.,adalimumab). In some embodiments, the gamma polyglutamated aminopterinis administered in combination with an fc-fusion protein (e.g.,entanercept).

In some embodiments, of the methods described herein, the combination ofan γPAMN compositions described herein and at least one additionaltherapeutic agent results in additive or synergistic results. In someembodiments, the combination therapy results in an increase in thetherapeutic index of the γPAMN or agent. In some embodiments, thecombination therapy results in an increase in the therapeutic index ofthe additional therapeutic agent(s). In some embodiments, thecombination therapy results in a decrease in the toxicity and/or sideeffects of the γPAMN or agent. In some embodiments, the combinationtherapy results in a decrease in the toxicity and/or side effects of theadditional therapeutic agent(s).

In some embodiments, in addition to administering gamma polyglutamatedAMN compositions described herein, the methods or treatments describedherein further comprise administering at least one additionaltherapeutic agent selected from: an anti-tubulin agent, an auristatin, aDNA minor groove binder, a DNA replication inhibitor, an alkylatingagent (e.g., platinum complexes such as cisplatin, mono(platinum),bis(platinum) and tri-nuclear platinum complexes and carboplatin), ananthracycline, an antibiotic, an anti-folate (e.g., a polyglutamatableantifolate or a non polyglutamatable anti-folate), an antimitotic (e.g.,a vinca alkaloid, such as vincristine, vinblastine, vinorelbine, orvindesine), radiation sensitizer, a steroid, a taxane, a topoisomeraseinhibitor (e.g., doxorubicin HCl, daunorubicin citrate, mitoxantroneHCl, actinomycin D, etoposide, topotecan HCl, teniposide (VM-26), andirinotecan), an anti-metabolite, a chemotherapy sensitizer, aduocarmycin, an etoposide, a fluorinated pyrimidine, an ionophore, alexitropsin, a nitrosourea, a platinol, a purine antimetabolite, a PARPinhibitor, and a puromycin. In certain embodiments, the secondtherapeutic agent is an alkylating agent, an antimetabolite, anantimitotic, a topoisomerase inhibitor, or an angiogenesis inhibitor.

Therapeutic agents that may be administered in combination with theγPAMN compositions described herein include chemotherapeutic agents.Thus, in some embodiments, the methods or treatments described hereinfurther comprise administering at least one involves the administrationof a γPAMN composition described herein in combination with achemotherapeutic agent or in combination with a cocktail ofchemotherapeutic agents. Treatment with a γPAMN composition can occurprior to, concurrently with, or subsequent to administration ofchemotherapies. Combined administration can include co-administration,either in a single pharmaceutical formulation or using separateformulations, or consecutive administration in either order butgenerally within a time period such that all active agents can exerttheir biological activities simultaneously. Preparation and dosingschedules for such chemotherapeutic agents can be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in The Chemotherapy Source Book, 4.sup.th Edition, 2008,M. C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, Pa.

Chemotherapeutic agents useful in the present invention include, but arenot limited to, alkylating agents such as thiotepa and cyclosphosphamide(CYTOXAN); alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaor-amide and trimethylolomelamime; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as aminopterin and5-fluorouracil (5-FU); folic acid analogues such as denopterin,aminopterin, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine,floxuridine, 5-FU; androgens such as calusterone, dromostanolonepropionate, epitiostanol, mepitiostane, testolactone; anti-adrenals suchas aminoglutethimide, mitotane, trilostane; folic acid replenishers suchas folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (Ara-C); taxoids, such as paclitaxel (TAXOL®) and docetaxel(TAXOTERE®); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; ibandronate; CPT11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine (XELODA); anti-hormonal agents such as, tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON);anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; and pharmaceutically acceptable salts, acids orderivatives of any of the above. In certain embodiments, the additionaltherapeutic agent is cisplatin. In certain embodiments, the additionaltherapeutic agent is carboplatin. In other embodiments, the additionaltherapeutic agent is oxaloplatin.

V. Kits Comprising γPAMN Compositions

The disclosure also provides kits that comprise the γPAMN compositionsdescribed herein and that can be used to perform the methods describedherein. In certain embodiments, a kit comprises at least one purifiedγPAMN composition in one or more containers.

In some embodiments the kits include a dosage amount (e.g., as used fortherapy or diagnosis) of at least one γPAMN compositions (e.g., a γPAMNliposome), or pharmaceutical formulation thereof, as disclosed herein.Kits may further comprise suitable packaging and/or instructions for useof the composition. Kits may also comprise a means for the delivery forthe composition, or pharmaceutical formulation thereof, such as asyringe for injection or other device as described herein and known tothose of skill in the art. One of skill in the art will readilyrecognize that the disclosed γPAMN compositions can be readilyincorporated into one of the established kit formats which are wellknown in the art.

Further provided are kits that comprise a γPAMN compositions as well asat least one additional therapeutic agent. In certain embodiments, thesecond (or more) therapeutic agent is an anti-metabolite. In certainembodiments, the second (or more) therapeutic agent is achemotherapeutic agent.

The following examples are intended to illustrate but not to limit thedisclosure in any manner, shape, or form, either explicitly orimplicitly. While they are typical of those that might be used, otherprocedures, methodologies, or techniques known to those skilled in theart may alternatively be used without departing from the scope of thepresent disclosure.

In some instances the antifolate pemetrexed is evaluated to determinethe effect of using a polyglutamated polyglutamatable antifolate. Theresult obtained for pemetrexed are expected to apply equally in kind(but not necessarily equally in magnitude) to other polyglutamatableantifolates such as aminopterin. Antifolates have been used andcharacterized in clinical and research settings for more than half acentury, and the role of polyglutamation upon polyglutamatableantifolates in impacting cellular cytotoxicity is well understood in theart.

FIG. 1B-1N shows chemical formulae of exemplary L-gamma polyglutamatedaminopterin compositions encompassed by the disclosure.

EXAMPLES Example 1: Liposomal Gamma Polyglutamated PemetrexedCompositions Methods: Production of Gamma Hexaglutamated Pemetrexed(γHgPMX) Liposomes

Briefly Gamma Hexaglutamated pemetrexed (gGR6) and D gammahexaglutamated pemetrexed (gDGR6) was encapsulated in liposomes by thefollowing procedure. First, the lipid components of the liposomemembrane were weighed out and combined as a concentrated solution inethanol at a temperature of around 65° C. In this example, the lipidsused were hydrogenated soy phosphatidylcholine, cholesterol, andDSPE-PEG-2000(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly-ethylene glycol)-2000]). The molar ratio ofHSPC:Cholesterol:PEG-DSPE was approximately 3:2:0.15. Next, the gGR6 orgDGR6 was dissolved in 5% dextrose at a concentration of 100-150 mg/mlwith a pH of 6.5-6.9. The drug solution was heated up to 65° C. Theethanolic lipid solution was injected into the gGR6 or gDGR6 solutionusing a small-bore needle. During this step the drug solution was wellstirred using a magnetic stirrer. The mixing was performed at anelevated temperature (63° C.-72° C.) to ensure that the lipids were inthe liquid crystalline state (as opposed to the gel state that theyattain at temperatures below the lipid transition temperature Tm=51°C.-54° C.). As a result, the lipids were hydrated and form multiplebilayer (multilamellar) vesicles (MLV) containing gGR6 or gDGR6 in theaqueous core.

Downsizing of MLV's Using Filter Extrusion

The MLVs were fragmented into unilamellar (single bilayer) vesicles ofthe desired size by high-pressure extrusion using three passes throughstacked (track-etched polycarbonate) membranes. The first pass wasperformed through stacked membranes consisting of two layers with a poresize of 200 nm. The remaining two passes were through the stackedmembranes consisting of three layers with a pore size of 100 nm. Duringextrusion, the temperature was maintained above the Tm to ensureplasticity of the lipid membranes. As a result of the extrusion, largeand heterogeneous in size and lamellarity MLVs turned into small,homogenous (90-125 nm) unilamellar vesicles (ULV) that sequestered thedrug in their interior. A Malvern Zetasizer Nano ZS instrument(Southborough, Mass.) with back scattering detector (90°) was used formeasuring the hydrodynamic size (diameter) at 25° C. in a quartz microcuvette. The samples were diluted 50-fold in formulation matrix beforeanalysis.

Purification of Liposomes

After the ULV's containing gGR6 or gDGR6 had been produced, theextra-liposomal free drug was removed using columns for small volume ortangential flow diafiltration against a suitable buffer for largevolume. Although any buffer solution can be used, in this example thebuffer used was 5 mM HEPES, 145 mM Sodium Chloride, pH 6.7. Uponcompletion of purification, filter sterilization was performed using a0.22 micron filter.

The typical characteristics of liposomal derivatives are shown in thetable below

Encapsulation Drug/Lipid Starting con. efficiency Final con. RatioDiameter PDI Zeta pot

Lps gDG6  1 mg/ml  5.71% 0.038 mg/ml 25-30 g/mM 103.8 nm 0.017 −1.77 m

lipids Lps gG6  20 mg/ml 10.60%  1.39 mg/ml 35-50 g/mM 114.9 nm 0.035−1.76 m

lipids Lps 100 mg/ml   34%  7.5 mg/ml 225-265 116.3 nm 0.045 −2.32 m

gG6 g/mM Lipids

indicates data missing or illegible when filed

Dose Response Study of Gamma HGP (Hexaglutamated Pemetrexed) andLiposomes

A dose response study was performed using liposomes containinghexaglutamated pemetrexed that were prepared essential as describedabove.

Cell viability was determined by CellTiter-Glo® (CTG) luminescent cellviability assay on Day 3 (48 hour) and Day 4 (72 hour). This assaydetermined the number of viable cells in culture based on quantifyingATP that was present within, which in turn signals the presence ofmetabolically active cells. The CTG assay uses luciferase as a readout.To assess cell viability Dose response inhibition of pemetrexed, HGP andliposomes on different cancer cell growth were investigated usingCellTiter-Glo® luminescent cell viability assay. Human cancer cells wereharvested, counted and plated at a same cell density on Day 0. A seriesof 8 dilutions of each test article were added to the cells on Day 1.Dose response curve were generated and fit using GraphPad Prism and IC50of each test article were calculated. A lower the IC50 is, the morepotent the test article was in term of cancer cell growth inhibition.

Cells were seeded into 96-well plate at a cell density of 5×10⁴ cellsper well in 100 μl of fresh media on Day 0. Eight serial 2-folddilutions of each test article in culture medium were generated andadded to cells in triplicate on Day 1. In addition, three wells of cellswere treated with vehicle (HBS for free drug or empty liposome forliposomal HGP) alone as a control.

On Days 3 and 4, 100 μl of CellTiterGlo® Reagent were added to each welland incubated at room temperature for 15 minutes. Luciferaseluminescence were recorded for each well. In addition, 8 serial 2-folddilutions of the vehicle (HBS or empty liposome) in culture medium wereadded into empty wells and included in the assay to generate thebackground luminescence signals. Luciferase signals were normalized bysubtracting the background luminescence signal out of the read-outsrespectively.

Human Normal Primary Bone Marrow CD34+ Cells were obtained from ATCC.(ATCC Catalog Number PCS-800-012). Cells were thawed at 37° C. for 1minute and then placed on ice. The cells were then resuspended inStemSpan SFEM (Stem Cell Tech Catalog Number 9650) plus 10% heatinactivated fetal bovine serum (Corning 35-015-CV). The cells wereplated into 96 well culture plates at a density of 2.5×10⁴ cells/well.The following day, live cells were collected via centrifugation andresuspended in neutrophil growth media (StemSpan SFEM plus 10% HeatInactivated fetal bovine serum plus 100 ng/ml human stem cell factor(Sigma Catalog Number H8416), 20 ng/ml human granulocytecolony-stimulation factor (Sigma Catalog Number H5541), and 10 ng/mlhuman recombinant IL3 (Sigma SRP3090) at a density of 2.5×10⁴cells/well. Cells were incubated at 37° C. for 10 days. Fresh media wasadded every two days. Mature neutrophils were then collected and platedin 96 well plates at a density of 1×10⁴ cells/well and incubated at 37°C. overnight. The next day, test article or vehicle was resuspended inneutrophil growth media and added to the plates. The cells were thenincubated for either 48 hours or 72 hours at 37° C. and then assayed ateach time point using the Cell Titer Glo Assay (Promega Catalog #G7572).

Methodologies used for cell line AML12 (non-cancerous liver cells) andCCD841 (non-cancerous colon epithelial cells) are similar to the methodsused for cancer cells.

Results

FIGS. 1B-1N show exemplary chemical formulae of gamma aminopterinpolyglutamates. FIG. 10 shows exemplary aminopterin molecules.

In a set of dose response experiments, 6 cell lines representingdifferent types of cancers, namely HT-29 (colon cancer), H2342 (NSCLC,adenocarcinoma subtype), H292 (NSCLC, adenocarcinoma subtype), SW620(CRC), H1806 (triple negative breast cancer) and OAW28 (ovarian cancer),were studied (FIG. 5). Treatment consisted of exposure for 48 hoursusing 2 different encapsulated derivatives of liposomal gamma pemetrexedhexaglutamate, namely liposomal gamma L hexaglutamate (liposomal gG6)and its mirror image, liposomal gamma D hexaglutamate (liposomal gDG6)also referred to as its corresponding enantiomer.

The relative potency of the above mentioned derivatives as compared topemetrexed, following exposure over 48 hours, is represented in FIG. 5.The relative potency of treatment using the various derivatives, asshown in this figure was calculated by dividing the IC50 of pemetrexedby the IC50 of the liposomal gamma pemetrexed hexaglutamate for eachcell line. As shown in this figure, in all cell lines, the potency ofliposomal gamma pemetrexed hexaglutamate well exceeded that ofpemetrexed. By way of example, consider the NSCLC cell line H292. Asshown in the figure, the potency of liposomal gamma pemetrexedhexaglutamate was ≥50-fold that of pemetrexed. This suggests that a 2%or lower dose of the liposomal gamma pemetrexed hexaglutamate could havethe same treatment effect as a 100% dose of pemetrexed.

Cancer cell viability studies comparing the liposomal gamma pemetrexedhexaglutamate derivatives (liposomal L gamma G6/Lps Hexa gG6 andliposomal D gamma G6/Lps Hexa gDG6) and pemetrexed for cytotoxicactivity on representative cell lines in breast, colon, lung and ovariancancer are shown in FIGS. 2, 3, 4, 6, 7 and 8. These data show that bothliposomal gamma L pemetrexed hexaglutamate and liposomal gamma Dpemetrexed hexaglutamate are more potent than pemetrexed. Further, as anindicator of efficacy, the results of the experiments on the same celllines depicted at various dose levels ranging from 16 to 128 nM in FIGS.9-11. As shown in these figures, at each of these dose ranges, liposomalgamma L pemetrexed hexaglutamate and liposomal gamma D pemetrexedhexaglutamate are superior to pemetrexed in terms of inhibiting cancercells for the lung and breast cancer cell lines. In the ovarian cancercell line, pemetrexed at the dose of 128 nM, appears to be equallyeffective as liposomal gamma pemetrexed hexaglutamate, whereas theliposomal gamma pemetrexed hexaglutamate at the dose of 32 nM and 64 nMhas a better treatment effect than pemetrexed; at 16 nM the treatmenteffect is lower and similar in magnitude for liposomal gamma pemetrexedhexaglutamate and pemetrexed.

The major toxicities seen in patients treated with pemetrexed is bonemarrow suppression which manifests as a decrease in blood countsincluding neutrophil counts (a type of white blood cells). There is alsosome adverse effect on the lining of the mouth and gut that manifests asdiarrhea and mucositis, as well as an adverse effect on the liver insome instances. To assess the above-mentioned toxicities, treatment ofthe liposomal gamma pemetrexed hexaglutamate derivatives (L and D) andpemetrexed was measured at 48 hours on CD34+ cells that weredifferentiated into neutrophils, CCD841 colon epithelium cells and AML12liver cells. As shown in FIG. 12, liposomal gamma pemetrexedhexaglutamate is significantly less toxic to differentiating humanneutrophils in contrast to pemetrexed. This is also supported byneutrophil counts that are better preserved following treatment with theliposomal gamma L pemetrexed hexaglutamate or liposomal gamma Dpemetrexed hexaglutamate compared to pemetrexed, at dose ranges from 16nM to 128 nM (FIG. 13). Strikingly, there does not appear to be anytoxicity to the liver cells following treatment with liposomal L gammapemetrexed hexaglutamate or liposomal gamma D pemetrexed hexaglutamateat the dose levels studied (FIG. 14). In contrast, pemetrexed at alldoses studied is leading to a reduction in the liver cell counts ofapproximately 40%. And finally, the same trend is seen followingtreatment of epithelial colon cells (FIG. 15). As shown in this figure,pemetrexed at all doses studied is leading to approximately a ≥50%decrease in the number of cells compared to approximately a 20% or lessdecrease after treatment with liposomal gamma L pemetrexed hexaglutamateand liposomal gamma D pemetrexed hexaglutamate.

Example 2: Targeted Liposome Polyglutamated Antifolate Cell DeliveryMethods Production of Targeted Gamma Hexaglutamated Pemetrexed (HGP)Liposomes

Gamma HGP (gG6) was encapsulated in liposomes and the liposomes weredownsized and purified according to procedures essentially as set forthabove in Example 1.

Antibody Conjugation

Activated liposomes were prepared by adding DSPE-PEG-maleimide to thelipid composition. The liposomes contain four different lipids:hydrogenated soy phosphatidylcholine (HSPC), cholesterol,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly ethyleneglycol)-2000] (DSPE-PEG-2000), and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG-maleimide), in ratios of3:2:0.1125:0.0375.

Antibody thiolation was accomplished through use of Traut's reagent(2-iminothiolane) to attach a sulfhydryl group onto primary amines.Antibody was suspended in PBS at a concentration of 0.9-1.6 mg/ml.Traut's reagent (14 mM) was added to antibody solution at a finalconcentration of 1-5 mM and then removed through dialysis after one-hourincubation at room temperature. Thiolated antibody was added toactivated liposome at a ratio of 60 g/mol phosphate lipids, and thereaction mixture was incubated for one hour at room temperature andover-night at 4 uL-cysteine was used to terminate the reaction andunconjugated antibody was removed through dialysis.

Exemplary direct and post insertion antibody-liposome conjugationmethods are provided below.

Exemplary Antibody Conjugation Method 1: Direct Conjugation

Antibody or its fragments, such as Fab or scFv, can be conjugateddirectly onto thiol-reactive liposome. Thiol-reactive liposomes areprepared by adding DSPE-PEG-maleimide to the lipid composition. Theliposomes contain four different lipids: hydrogenated soyphosphatidylcholine (HSPC), cholesterol,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly ethyleneglycol)-2000] (DSPE-PEG-2000), and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG-maleimide), in ratios of3:2:0.1125:0.0375.

Antibody (or its fragments, such as Fab or scFv) thiolation isaccomplished through use of Traut's reagent (2-iminothiolane) to attacha sulfhydryl group onto primary amines. Antibody (or its fragment) issuspended in PBS at a concentration of 0.9-1.6 mg/ml. Traut's reagent(14 mM) is added to antibody (or its fragment) solution at a finalconcentration of 1-5 mM and then removed through dialysis after one-hourincubation at room temperature. Thiolated antibody (or its fragment) isadded to thiol-reactive liposome at a ratio of 60 g/mol phosphatelipids, and the reaction mixture is incubated for one hour at roomtemperature and over-night at 4° C. L-cysteine is used to terminate thereaction and unconjugated antibody (or its fragment) is removed throughdialysis.

Antibody or its fragments, such as Fab or scFv, which contains acysteine residue at the C-terminal can be conjugated directly onto theliposome by incubating a reduced antibody (or its fragment) withthiol-reactive liposome. Antibody (or its fragment) with a cysteine tailis dissolved and reduced by a 10-20 mM reducing reagent (such as2-mercaptoethylamine, cysteine, or dithioerythritol) at pH<7. The excessreducing reagent is removed thoroughly by size exclusion chromatographyor dialysis. The purified and reduced antibody (or its fragment) can bedirectly conjugated to the thiol-reactive liposome.

Exemplary Antibody Conjugation Method 2: Post Insertion

Antibody or its fragments, such as Fab or scFv, which contains acysteine residue at the C-terminal can be conjugated and incorporatedinto the liposome through a “post insertion” method. Micelles ofthiol-reactive lipopolymer (such as DSPE-PEG-maleimide) is prepared bydissolving in an aqueous solution at 10 mg/ml. Antibody (or itsfragment) with a cysteine tail is dissolved and reduced by a 10-20 mMreducing reagent (such as 2-mercaptoethylamine, cysteine, ordithioerythritol) at pH<7. The excess reducing reagent is removedthoroughly by size exclusion chromatography or dialysis. The purifiedand reduced antibody (or its fragment) is then incubated with themicelles of thiol-reactive lipopolymers at a molar ratio of 1:4. At theend of the reaction, the excess maleimide groups are quenched by a smallamount of cysteine (1 mM) or mercaptoethanol. Unconjugated antibody (orits fragment) is removed by size exclusion chromatography. Purifiedconjugated micelles is then incubated with liposome at 37° C. orelevated temperature.

Physical Characteristics of the Nanoparticles Dose Response Study of HGP(Pentaglutamated Pemetrexed) and Liposomes.

Cell viability was determined by CellTiter-Glo® (CTG) luminescent cellviability assay on Day 3 (48 hour) and Day 4 (72 hour). This assaydetermines the number of viable cells in culture based on quantifyingATP that was present within, which in turn signals the presence ofmetabolically active cells. The CTG assay uses luciferase as a readout.To assess cell viability Dose response inhibition of pemetrexed, HGP andliposomes on different cancer cell growth were investigated usingCellTiter-Glo® luminescent cell viability assay. Human cancer cells wereharvested, counted and plated at a same cell density on Day 0. A seriesof 8 dilutions of each test article were added to the cells on Day 1.Dose response curve were generated and fit using GraphPad Prism and IC50of each test article were calculated. A lower the IC50 is, the morepotent the test article was in term of cancer cell growth inhibition.

Cells were seeded into 96-well plate at a cell density of 5×10⁴ cellsper well in 100 μl of fresh media on Day 0. Eight serial 2-folddilutions of each test article in culture medium were generated andadded to cells in triplicate on Day 1. In addition, three wells of cellswere treated with vehicle (HBS for free drug or empty liposome forliposomal HGP) alone as a control.

On Days 3 and 4, 100 μl of CellTiterGlo® Reagent were added to each welland incubated at room temperature for 15 minutes. Luciferaseluminescence were recorded for each well. In addition, 8 serial 2-folddilutions of the vehicle (HBS or empty liposome) in culture medium wereadded into empty wells and included in the assay to generate thebackground luminescence signals. Luciferase signals were normalized bysubtracting the background luminescence signal out of the read-outsrespectively.

Human Normal Primary Bone Marrow CD34+ Cells were obtained from ATCC.(ATCC Catalog Number PCS-800-012). Cells were thawed at 37° C. for 1minute and then placed on ice. The cells were then resuspended inStemSpan SFEM (Stem Cell Tech Catalog Number 9650) plus 10% heatinactivated fetal bovine serum (Corning 35-015-CV). The cells wereplated into 96 well culture plates at a density of 2.5×10⁴ cells/well.The following day, live cells were collected via centrifugation andresuspended in neutrophil growth media (StemSpan SFEM plus 10% HeatInactivated fetal bovine serum plus 100 ng/ml human stem cell factor(Sigma Catalog Number H8416), 20 ng/ml human granulocytecolony-stimulation factor (Sigma Catalog Number H5541), and long/mlhuman recombinant IL3 (Sigma SRP3090) at a density of 2.5×10⁴cells/well. Cells were incubated at 37° C. for 10 days. Fresh media wasadded every two days. Mature neutrophils were then collected and platedin 96 well plates at a density of 1×10⁴ cells/well and incubated at 37°C. overnight. The next day, test article or vehicle was resuspended inneutrophil growth media and added to the plates. The cells were thenincubated for either 48 hours or 72 hours at 37° C. and then assayed ateach time point using the Cell Titer Glo Assay (Promega Catalog #G7572).

Methodologies used for cell line AML12 (non-cancerous liver cells) andCCD841 (non-cancerous colon epithelial cells) are similar to the methodsused for cancer cells.

Results:

The dose response relationship of free pemetrexed gamma hexaglutamate(gG6), (non-targeted) liposomal gamma hexaglutamate (liposomal gG6),pemetrexed and folate receptor alpha targeting antibody (FR1Ab)liposomal pemetrexed gamma hexaglutamate (liposomal gG6-FR1Ab), in theNCI H2342 non-small cell lung cancer (NSCLC), adenocarcinoma subtype isshown in FIG. 2. The output is percentage of viable cells after 48 hoursof treatment as measured by luciferase luminescence. As shown in FIG. 2,the free pemetrexed gG6 appears to be the least potent as measured byIC50. Both the liposomal pemetrexed gG6 and the liposomal pemetrexedgG6-FR1Ab are 7-fold and 40-fold more potent, respectively, than freepemetrexed.

Similar data is shown for the HT-29 colon cancer cell line in FIG. 3that depict cell viability expressed as a percentage. As shown in thisfigure, free pemetrexed gG6 appears to be the least potent. In thisinstance, the liposomal pemetrexed gG6 is twice as potent as pemetrexedand the liposomal pemetrexed gG6-FR1Ab is 5-fold more potent than freepemetrexed.

Example 3: In Vivo Studies

The following example describes in vivo efficacy and toxicity dataobtained upon administering alpha G6 (Lp-aG6) (alpha polyglutamatedpemetrexed) in an in-vivo (murine) model. Those skilled in the art willappreciate that the efficacy and reduced toxicity observed for liposomalalpha polyglutamated pemetrexed compositions is expected to also beobserved upon administration of the counterpart liposomal gammapolyglutamated pemetrexed (gamma G6 (Lp-gG6) under the same conditions,albeit at possibly different levels.

Methods: Safety Studies in Mice

Because some of the major toxicities associated with a pemetrexed basedtreatment are hematologic and hepatic, it is important to evaluate theeffect of Liposomal alpha G6 (Lp-aG6) in an in-vivo (murine) model andcompare the changes in hematologic and the liver serum chemistry panelfollowing treatment. To obtain this data an initial dose ranging studywas conducted using healthy female BALB/c mice (6-8 weeks old) whichwere purchased from The Jackson Laboratory (Bar Harbor, Me.). Prior tothe study, animals were weighed, randomized by weight, observed forclinical abnormalities, and distributed into groups (5 mice per group).Doses from 10 mg/kg up to 200 mg/kg were investigated to identify atolerable dose in mice. Treatments were administrated intravenously oncea week for four weeks. Body weight and detailed clinical observationwere recorded daily. At the end of study, Day 28, mice were euthanized,and blood and tissue were harvested from untreated Control mice and forthe mice treated with Liposomal aG6 (Lp-aG6) 40 mg/kg and Liposomal aG680 (Lp-aG6) mg/kg. Whole blood was collected into K2-EDTA anticoagulanttubes for comprehensive complete blood count (CBC) and serum wasisolated for comprehensive chemistry and was sent to IDEXX (Westbrook,Me.) on the day of collection.

Results:

In general, treatment with once weekly liposomal aG6 at two dose levelsof 40 mg/kg and 80 mg/kg for 4 weeks was well tolerated and there wereno major differences in weight compared to untreated controls. To assesssome of the effects on hematologic parameters, white blood cell (WBC)counts, neutrophil counts as well as platelet counts were measured aftertreatment with liposomal aG6 at two dose levels of 40 mg/kg and 80 mg/kgboth given once weekly for 4 weeks. As can be seen in FIG. 17, therewere no appreciable decreases in mean neutrophil, mean white blood celland mean platelet counts, after four weeks of treatment with LiposomalaG6 in treated animals compared to untreated control animals. Hemoglobinand reticulocyte indices were measured to assess the impact on red bloodcell. As shown in FIG. 18, there was a minimal decrease in meanhemoglobin concentrations at the higher dose level. In parallel there isa slight increase in mean reticulocytosis indices which suggests a bonemarrow's response to treatment by increasing red blood cell production.Altogether this effect seems minor as the mice hemoglobin levels aremaintained after 4 weeks of treatment. Taken together these data suggestthat at these dose levels, 40 mg/kg and 80 mg/kg once-weekly, there islittle impact on the bone marrow and related hematologic indices.

Another concern with pemetrexed is hepatic toxicity that has beenobserved in some patients treated with pemetrexed based therapy. Toassess hepatic well being in mice serum chemistries including serumaspartate transaminase (AST) and serum alanine transaminase (ALT) alongwith serum albumin were measured. As shown in FIG. 19, there were noappreciable increases in liver transaminases mean AST and mean ALTlevels at 4 weeks following treatment with Liposomal aG6 at the two doselevels of 40 mg/kg and 80 mg/kg both given once weekly for 4 weeks whencompared to untreated controls. There was no change in mean albuminlevels either. Taken together these data suggest a favorable safetyprofile for Liposomal aG6.

Preliminary Pilot Efficacy Study in Mice Xenografts

To assess whether there was any tumor control following treatment withLiposomal alpha G6 (Lp-aG6) the pilot study was conducted. In this studyimmunodeficient female Nude mice (Nu/J; 6-8 weeks old) were purchasedfrom The Jackson Laboratory (Bar Harbor, Me.). NCI-H292 (Non-Small CellLung Cancer) cells were cultured in RPMI media supplemented with 10%Fetal Bovine Serum in a 37° C., 5% CO₂ incubator. 1×10⁶ cells wereinoculated subcutaneously into the dorsal hind flank of each mouse.Tumor volume and body weight were monitored twice every week.Tumor-bearing mice were randomized by tumor volume on Day 0 anddistributed into groups (5 mice per group): Control, Pemetrexed, andLiposomal aG6. Pemetrexed was given intravenously at 167 mg/kg onceevery three weeks. This murine dose of 167 mg/kg every three weeks isequivalent to the FDA/EMA approved human dose and schedule of 500 mg/m²every three weeks. Liposomal aG6 was dosed intravenously at 80 mg/kgonce a week for four weeks. Tumor size was measured with a caliper andtumor burden is calculated using the following equations: tumorvolume=0.5×(tumor length)×(tumor width)²; Relative tumor volume=(tumorvolume/tumor volume on Day 0)×100%. This study is still ongoing butpreliminary data are shown in FIG. 20. In this figure, relative tumorvolume is displayed following treatment with Liposomal aG6 andpemetrexed. As can be seen from these preliminary data, liposomal aG6provides better tumor control when compared to pemetrexed.

Example 4: Polyglutamated Antifolate-Cisplatin Complexes (PGPD) Methods:

Folate Analogues also known as antifolate have been an importantanticancer treatment for the last 70 years. Used in this setting thisclass of anti-cancer drugs interferes with various enzymes in theimportant folate metabolic pathway. This can result in impairedpyrimidine and purine (DNA and RNA) synthesis, impaired amino acidglycine and serine metabolism, impaired redox response and impairedmethylation processes within the cell.

In clinical practice, antifolates such as pemetrexed and aminopterin areoften used in combination with platinum agents such as cisplatin andcarboplatin. The combinations result in enhanced efficacy. In thiscontext, we set out to coencapsulated the polyglutamates with platinumagents in a specific ratio to facilitate controlled delivery of apredetermined ratio of the two anticancer drugs namely a polyglutamatedantifolate and a platinum analogue. We surprisingly discovered that longforms of polyglutamate antifolate (e.g., pentaglutamated antifolate)forms a complex with cisplatin that is stable at high pH, and that thiscomplex disassociates into polyglutamate and cisplatin at low pH. Low pHis believed to be occur in many tumor cells and the tumor cellenvironment, particularly in hypoxic settings. Application of thisdiscovery provides the ability to facilitate the delivery ofcombinations of γPPMX and therapeutic agents such as cisplatin to targetcells such as tumor cells and to release the drugs from the complex inphysiologically relevant low pH conditions. Production of Polyglutamatedantifolates—DDAP (Cisplatin) Complexes (PGPD)

To produce (Polyglutamated antifolates—DDAP Complex), alphahexaglutamate (aG6) and Diammine dicarboxylic acid platinum (DDAP) wasused. The process of complexation was dependent on the presence ofChlorinated platinum compound and pH conditions. The complexation wasachieved by a nucleophilic attack on one or two carboxyl groups ofglutamate by the platinate derivative. Briefly the complex was formed bythe following procedure. First, the active compound DDAP was weighed anddissolved in in 5% dextrose. After the DDAP dissolution step, aG6 wasweighed out and added to the DDAP-Captisol solution and allowed to stirfor 1 hour at 45-55° C. The pH of the solution was adjusted to 6.5-7.0using 1N NaOH and the solution was stirred for 1-2 hour. The formationof complex was confirmed visually. However, when the pH is adjusted toacidic pH 3 to 5, the color reverted back to its original, indicatingthe decomplexation of the polyglutmated antifolate and cisplatin.

Complex formation was confirmed using HPLC which showed two distinctpeaks that merge into 1 large peak at high pH of 6.5 to 7.5 and thenreappear at low pH of 3 to 5. Repeating the experiment without Captisolshowed that complex formation was independent of Captisol®. FIG. 16depicts structure of polyglutamate antifolate, cisplatin (CDDP) and twopotential gG6-Cisplatin complexes. The pH dependent formation of theinterstrand and/or instrastrand coordination between the carboxyl groupsof the polyglutamated antifolate and cisplatin is likely to disassembleinto individual molecules of gG6 and cisplatin upon encountering acidicpH of lysosomes (pH 3-5) and presence of chloride ions inside the cells.Production of Pentaglutamated Pemetrexed-DDAP/CDDP complex (PGPD)Liposomes

Briefly PGPD was encapsulated in liposomes by the following procedure.First, the lipid components of the liposome membrane was weighed out andcombined as a concentrated solution in ethanol at a temperature ofaround 65° C. In this example, the lipids used were hydrogenated soyphosphatidylcholine, cholesterol, and DSPE-PEG-2000(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]). The molar ratio of HSPC:Cholesterol:PEG-DSPE wasapproximately 3:2:0.15. Next, PGPD was prepared as described above. ThePGPD drug solution was heated up to 65° C. The ethanolic lipid solutionwas injected into the PGPD solution using a small-bore needle. Duringthis step the drug solution was well stirred using a magnetic stirrer.The mixing was performed at an elevated temperature (63° C.-72° C.) toensure that the lipids were in the liquid crystalline state (as opposedto the gel state that they attain at temperatures below the lipidtransition temperature Tm=51° C.-54° C.). As a result, the lipids werehydrated and formed multiple bilayer (multilamellar) vesicles (MLV)containing PGPD in the aqueous core.

Downsizing of MLV's Using Filter Extrusion

The MLVs were fragmented into unilamellar (single bilayer) vesicles ofthe desired size by high-pressure extrusion using two passes throughstacked (track-etched polycarbonate) membranes. The stacked membraneshave two layers with a pore size of 200 nm and six layers with a poresize of 100 nm. During extrusion, the temperature was maintained abovethe Tm to ensure plasticity of the lipid membranes. Because of theextrusion, large and heterogeneous in size and lamellarity MLVs turninto small, homogenous (90-120 nm) unilamellar vesicles (ULV) thatsequester the drug in their interior. A Malvern Zetasizer Nano ZSinstrument (Southborough, Mass.) with back scattering detector (90°) wasused for measuring the hydrodynamic size (diameter) at 25° C. in apolystyrene micro cuvette. The samples were diluted 50-fold informulation matrix before analysis.

Purification of Liposomes:

After the ULV's containing PGPD had been produced, the extra-liposomalPGPD was removed using columns for small volume or tangential flowdiafiltration against a suitable buffer for large volume. Although manydifferent buffers known in the art could have been used, in this examplethe buffer used was 5 mM HEPES, 145 mM Sodium Chloride, pH 6.7. Uponcompletion of purification, filter sterilization was performed using a0.22-micron filter.

FURTHER EMBODIMENTS

In a non-limiting embodiment, of this disclosure, there is provided acomposition comprising gamma polyglutamated aminopterin.

In the composition of the immediately preceding paragraph, thecomposition may comprise pentaglutamated or hexaglutamated aminopterin.

In the composition of any of the preceding two paragraphs, thecomposition may comprise gamma polyglutamated aminopterin which mayinclude pentaglutamated or hexaglutamated aminopterin.

A non-limiting example liposomal gamma polyglutamated aminopterin(L-γPAMN) composition may comprise a composition of any of the precedingthree paragraphs and the liposome may be optionally pegylated(PL-γPAMN).

In the L-γPAMN or PL-γPAMN composition of the immediately precedingparagraph, the gamma polyglutamated aminopterin may includepentaglutamated or hexaglutamated aminopterin.

In the L-γPAMN or PL-γPAMN composition of any of the preceding twoparagraphs, the liposome may be anionic or neutral.

In the L-γPAMN or PL-γPAMN composition of any of the preceding threeparagraphs, a targeting moiety may be attached to one or both of a PEGand the exterior of the liposome, and the targeting moiety may have aspecific affinity for a surface antigen on a target cell of interest(TL-γPAMN or TPL-γPAMN).

In the L-γPAMN or PL-γPAMN composition of any of the preceding fourparagraphs, a targeting moiety may be attached to one or both of a PEGand the exterior of the liposome and may be a polypeptide.

In the L-γPAMN or PL-γPAMN composition of any of the preceding fiveparagraphs, a targeting moiety may be attached to one or both a PEG andthe exterior of the liposome and may be an antibody or a fragment of anantibody.

In the L-γPAMN or PL-γPAMN composition of any of the preceding sixparagraphs, one or more of an immunostimulatory agent, a detectablemarker and a maleimide may be disposed on at least one of a PEG and theexterior of the liposome.

In the L-γPAMN or PL-γPAMN composition of any of the preceding sevenparagraphs, a polypeptide may bind an antigen with an equilibriumdissociation constant (Kd) in a range of 0.5×10⁻¹⁰ to 10×10⁻⁶ asdetermined using BIACORE analysis.

In the L-γPAMN or PL-γPAMN composition of any of the preceding eightparagraphs, a polypeptide may specifically bind one or more folatereceptors selected from the group consisting of: folate receptor alpha(FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ).

A non-limiting exemplary method of killing a hyperproliferative cellthat includes contacting a hyperproliferative cell with a liposomalgamma polyglutamated aminopterin composition of any of the precedingnine paragraphs.

In the method of the immediately preceding paragraph, thehyperproliferative cell is a cancer cell.

A non-limiting example method for treating cancer comprisesadministering an effective amount of the gamma polyglutamatedaminopterin composition of any of preceding paragraphs from precedingparagraph eleven to preceding paragraph three, to a subject having or atrisk of having cancer.

In the method of the immediately preceding paragraph, the cancer may beone or more selected from the group consisting of: lung cancer,pancreatic, breast cancer, ovarian cancer, lung cancer, prostate cancer,head and neck cancer, gastric cancer, gastrointestinal cancer, coloncancer, esophageal cancer, cervical cancer, kidney cancer, biliary ductcancer, gallbladder cancer, and a hematologic malignancy.

A non-limiting example maintenance therapy for subjects that areundergoing or have undergone cancer therapy includes administering aneffective amount of the gamma polyglutamated aminopterin composition ofany of preceding paragraphs from preceding paragraph thirteen topreceding paragraph five, to a subject that is undergoing or hasundergone cancer therapy.

A non-limiting example pharmaceutical composition may include any gammapolyglutamated aminopterin composition of Section IV.

A non-limiting example method for treating a disorder of the immunesystem may include administering an effective amount of the of the gammapolyglutamated aminopterin composition of any of preceding paragraphsfrom preceding paragraph fourteen to preceding paragraph six, to asubject having or at risk of having a disorder of the immune system.

A non-limiting example method for treating an infectious may includecomprises administering an effective amount of the of the gammapolyglutamated aminopterin composition of any of preceding paragraphsfrom preceding paragraph fifteen to preceding paragraph seven, to asubject having or at risk of having an infectious disease.

A non-limiting example method of delivering gamma polyglutamatedaminopterin to a tumor expressing a folate receptor on its surface mayinclude administering a polyglutamated aminopterin composition of any ofpreceding paragraphs from preceding paragraph sixteen to precedingparagraph eight, to a subject having the tumor in an amount to deliver atherapeutically effective dose of the gamma polyglutamated aminopterinto the tumor.

A non-limiting example method of preparing a liposomal gammapolyglutamated aminopterin composition which includes gammapolyglutamated aminopterin composition of any of preceding paragraphsfrom preceding paragraph seventeen to preceding paragraph nine includesforming a mixture comprising: liposomal components; gamma polyglutamatedaminopterin in solution; homogenizing the mixture to form liposomes inthe solution; and processing the mixture to form liposomes containingthe polyglutamated aminopterin.

A non-limiting example pharmaceutical composition includes a gammapolyglutamated aminopterin composition of any of preceding paragraphsfrom preceding paragraph eighteen to preceding paragraph ten.

Although the disclosure has been described with reference to varioussome embodiments, it should be understood that various modifications canbe made without departing from the spirit of the disclosure.Accordingly, the scope of the disclosure should be determined withreference to the appended claims, along with the full scope ofequivalents to which such claims are entitled. Throughout thisapplication, various publications are referenced by author name anddate, or by Patent No. or Patent Publication No. The disclosure of thesepublications are hereby incorporated in their entireties by referenceinto this application in order to more fully describe the state of theart as known to those skilled therein as of the date of the inventiondescribed and claimed herein. However, the citation of a referenceherein should not be construed as an acknowledgement that such referenceis prior art to the present invention.

Various new chemical entities, methods and equipment for making thesechemical entities are set forth below in the appended claims. It is tobe appreciated that the Detailed Description section, and not theSummary and Abstract sections, is intended to be used to interpret theclaims. The Summary and Abstract sections may set forth one or more butnot all exemplary embodiments, of the present invention as contemplatedby the inventor(s), and thus, are not intended to limit the presentinvention and the appended claims in any way.

The disclosure of each of U.S. Appl. No. 62/627,732, filed Feb. 7, 2018;U.S. Appl. No. 62/627,733, filed Feb. 7, 2018; U.S. Appl. No.62/630,613, filed Feb. 14, 2018; U.S. Appl. No. 62/630,620, filed Feb.14, 2018; U.S. Appl. No. 62/630,625, filed Feb. 14, 2018; U.S. Appl. No.62/630,652, filed Feb. 14, 2018; U.S. Appl. No. 62/630,751, filed Feb.14, 2018; U.S. Appl. No. 62/630,824, filed Feb. 14, 2018; U.S. Appl. No.62/636,289, filed Feb. 28, 2018; U.S. Appl. No. 62/662,372, filed Apr.25, 2018; U.S. Appl. No. 62/702,774, filed Jul. 24, 2018; U.S. Appl. No.62/702,779, filed Jul. 24, 2018; U.S. Appl. No. 62/764,945, filed Aug.17, 2018; and U.S. Appl. No. 62/764,951, filed Aug. 17, 2018; is hereinincorporated by reference in its entirety.

What is claimed is:
 1. A composition comprising a gamma polyglutamatedaminopterin.
 2. The composition of claim 1, wherein the gammapolyglutamated aminopterin comprises 1-10 glutamyl groups having gammacarboxyl group linkages.
 3. The composition of claim 1 or 2, wherein thegamma polyglutamated aminopterin contains 4, 5, 2-10, 4-6, or more than5, glutamyl groups having gamma carboxyl group linkages.
 4. Thecomposition according to any of claims 1-3, wherein the gammapolyglutamated aminopterin is gamma tetraglutamated aminopterin.
 5. Thecomposition according to any of claims 1-3, wherein the gammapolyglutamated aminopterin is gamma pentaglutamated aminopterin.
 6. Thecomposition according to any of claims 1-3, wherein the gammapolyglutamated aminopterin is gamma hexaglutamated aminopterin.
 7. Thecomposition according to any of claims 1-6, wherein (a) the gammapolyglutamated aminopterin comprises two or more glutamyl groups in theL-form having gamma carboxyl group linkages, (b) each of the glutamylgroups of the gamma polyglutamated aminopterin is in the L-form and hasa gamma carboxyl group linkage, (c) at least one of the glutamyl groupsof the gamma polyglutamated aminopterin is in the D-form and has a gammacarboxyl group linkage, (d) each of the glutamyl groups of the gammapolyglutamated aminopterin other than the glutamyl group of aminopterinis in the D-form and has a gamma carboxyl group linkage, or (e) thegamma polyglutamated aminopterin comprises two or more glutamyl groupsin the L-form and at least one glutamyl group in the D-form having gammacarboxyl group linkages.
 8. The composition according to 4, wherein (a)each of the glutamyl groups is in the L-form and has a gamma carboxylgroup linkage or (b) each of the glutamyl groups other than the glutamylgroup of aminopterin is in the D-form and each of the glutamyl groupshas a gamma carboxyl group linkage.
 9. The composition of claim 5,wherein (a) each of the glutamyl groups is in the L-form and has a gammacarboxyl group linkage or (b) each of the glutamyl groups other than theglutamyl group of aminopterin is in the D-form and each of the glutamylgroups has a gamma carboxyl group linkage.
 10. The composition of claim6, wherein (a) each of the glutamyl groups is in the L-form and has agamma carboxyl group linkage or (b) each of the glutamyl groups otherthan the glutamyl group of aminopterin is in the D-form and each of theglutamyl groups has a gamma carboxyl group linkage.
 11. The compositionaccording to any of claims 1-10, wherein the gamma polyglutamatedaminopterin is polyglutamable by FGPS under normal physiologicconditions and/or wherein the polyglutamated AMN has a lower uptake rate(<30%) by hepatic cells than AMN.
 12. A liposomal composition comprisingthe gamma polyglutamated aminopterin according to any of claims 1-11(Lp-γPAMN).
 13. The Lp-γPAMN composition according to 12, wherein thegamma polyglutamated aminopterin comprises two or more glutamyl groupsin the L-form.
 14. The Lp-γPAMN composition according to 12 or 13,wherein each of the glutamyl groups of the gamma polyglutamatedaminopterin is in the L-form.
 15. The Lp-γPAMN composition of claim 12or 13, wherein at least one of the glutamyl groups of the gammapolyglutamated aminopterin is in the D-form.
 16. The Lp-γPAMNcomposition according to any of claims 12-15, wherein the liposomecomprises a gamma polyglutamated aminopterin comprising 1-10 glutamylgroups having gamma carboxyl group linkages.
 17. The Lp-γPAMNcomposition according to any of claims 12-16, wherein the liposomecomprises a gamma polyglutamated aminopterin containing 4, 5, 2-10, 4-6,or more than 5, glutamyl groups.
 18. The Lp-γPAMN composition accordingto any of claims 12-17, wherein the liposome comprises gammatetraglutamated aminopterin.
 19. The Lp-γPAMN composition according toany of claims 12-17, wherein the liposome comprises gammapentaglutamated aminopterin.
 20. The Lp-γPAMN composition according toany of claims 12-17, wherein the liposome comprises gamma hexaglutamatedaminopterin.
 21. The Lp-γPAMN composition according to any of claims12-20, wherein the liposome is not pegylated (PγLp-γPAMN).
 22. TheLp-γPAMN composition according to any of claims 12-20, wherein theliposome is pegylated (PγLp-γPAMN).
 23. The Lp-γPAMN compositionaccording to any of claims 12-23, wherein the liposomes comprise atleast 1% weight by weight (w/w) of the gamma polyglutamated aminopterinor wherein during the process of preparing the Lp-γPAMN, at least 1% ofthe starting material of gamma polyglutamated AMN is encapsulated(entrapped) in the Lp-γPAMN.
 24. The Lp-γPAMN composition according toany of claims 12-24, wherein the liposome has a diameter in the range of20 nm to 500 nm.
 25. The Lp-γPAMN composition according to any of claims12-24, wherein the liposome has a diameter in the range of 20 nm to 200nm.
 26. The Lp-γPAMN composition according to any of claims 12-25,wherein the liposome has a diameter in the range of 80 nm to 120 nm. 27.The Lp-γPAMN composition according to any of claims 12-26, wherein theliposome is formed from liposomal components.
 28. The Lp-γPAMNcomposition according to 27, wherein the liposomal components compriseat least one of an anionic lipid and a neutral lipid.
 29. The Lp-γPAMNcomposition according to 27 or 28, wherein the liposomal componentscomprise at least one selected from the group consisting of: DSPE;DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol;cholesterol-PEG; and cholesterol-maleimide.
 30. The Lp-γPAMN compositionaccording to any of claims 27-29, wherein the liposomal componentscomprise at least one selected from the group consisting of: DSPE;DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC. 31.The Lp-γPAMN composition according to any of claims 27-30, wherein oneor more liposomal components further comprises a steric stabilizer. 32.The Lp-γPAMN composition according to 31, wherein the steric stabilizeris at least one selected from the group consisting of polyethyleneglycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1);poly(vinyl pyrrolidone) (PVP); poly(acrylamide) (PAA);poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidylpolyglycerol; poly[N-(2-hydroxypropyl) methacrylamide]; amphiphilicpoly-N-vinylpyrrolidones; L-amino-acid-based polymer; oligoglycerol,copolymer containing polyethylene glycol and polypropylene oxide,Poloxamer 188, and polyvinyl alcohol.
 33. The Lp-γPAMN compositionaccording to 32, wherein the steric stabilizer is PEG and the PEG has anumber average molecular weight (Mn) of 200 to 5000 daltons.
 34. TheLp-γPAMN composition according to any of claims 12-33, wherein theliposome is anionic or neutral.
 35. The Lp-γPAMN composition accordingto any of claims 12-33, wherein the liposome has a zeta potential thatis less than or equal to zero.
 36. The Lp-γPAMN composition according toany of claims 12-33, wherein the liposome has a zeta potential that isbetween 0 to −150 mV.
 37. The Lp-γPAMN composition according to any ofclaims 12-33, wherein the liposome has a zeta potential that is between−30 to −50 mV.
 38. The Lp-γPAMN composition according to any of claims12-33, wherein the liposome is cationic.
 39. The Lp-γPAMN compositionaccording to any of claims 12-38, wherein the liposome has an interiorspace comprising the gamma polyglutamated aminopterin and an aqueouspharmaceutically acceptable carrier.
 40. The Lp-γPAMN composition ofclaim 39, wherein the pharmaceutically acceptable carrier comprises atonicity agent such as dextrose, mannitol, glycerine, potassiumchloride, sodium chloride, at a concentration of greater than 1%. 41.The Lp-γPAMN composition of claim 39, wherein the aqueouspharmaceutically acceptable carrier is trehalose.
 42. The Lp-γPAMNcomposition of claim 41, wherein the pharmaceutically acceptable carriercomprises 1% to 50% trehalose.
 43. The Lp-γPAMN composition according toany of claims 39-42, wherein the pharmaceutically acceptable carriercomprises 1% to 50% dextrose solution.
 44. The Lp-γPAMN compositionaccording to any of claims 39-43, wherein the interior space of theliposome comprises 5% dextrose suspended in an HEPES buffered solution.45. The Lp-γPAMN composition according to any of claims 39-44, whereinthe pharmaceutically acceptable carrier comprises a buffer such as HEPESBuffered Saline (HBS) or similar, at a concentration of between 1 to 200mM and a pH of between 2 to
 8. 46. The Lp-γPAMN composition according toany of claims 39-45, wherein the pharmaceutically acceptable carriercomprises a total concentration of sodium acetate and calcium acetate ofbetween 50 mM to 500 mM.
 47. The Lp-γPAMN composition according to anyof claims 12-46, wherein the interior space of the liposome has a pH of5-8 or a pH of 6-7, or any range therein between.
 48. The Lp-γPAMNcomposition according to any of claims 12-47, wherein the liposomecomprises less than 500,000 or less than 200,000 molecules of the gammapolyglutamated aminopterin.
 49. The Lp-γPAMN composition according toany of claims 12-48, wherein the liposome comprises between 10 to100,000 molecules of the gamma polyglutamated aminopterin, or any rangetherein between.
 50. The Lp-γPAMN composition according to any of claims12-49, which further comprises a targeting moiety and wherein thetargeting moiety has a specific affinity for a surface antigen on atarget cell of interest.
 51. The Lp-γPAMN composition according to 50,wherein the targeting moiety is attached to one or both of a PEG and theexterior of the liposome, optionally wherein targeting moiety isattached to one or both of the PEG and the exterior of the liposome by acovalent bond.
 52. The Lp-γPAMN composition of claim 50 or 51, whereinthe targeting moiety is a polypeptide.
 53. The Lp-γPAMN compositionaccording to any of claims 50-52, wherein the targeting moiety is anantibody or an antigen binding fragment of an antibody.
 54. The Lp-γPAMNcomposition according to any of claims 50-53, wherein the targetingmoiety binds the surface antigen with an equilibrium dissociationconstant (Kd) in a range of 0.5×10⁻¹⁰ to 10×10⁻⁶ as determined usingBIACORE analysis.
 55. The Lp-γPAMN composition according to any ofclaims 50-54, wherein the targeting moiety specifically binds one ormore folate receptors selected from the group consisting of: folatereceptor alpha (FR-α), folate receptor beta (FR-β), and folate receptordelta (FR-δ).
 56. The Lp-γPAMN composition according to any of claims50-55, wherein the targeting moiety comprises one or more selected fromthe group consisting of: an antibody, a humanized antibody, an antigenbinding fragment of an antibody, a single chain antibody, asingle-domain antibody, a bi-specific antibody, a synthetic antibody, apegylated antibody, and a multimeric antibody.
 57. The Lp-γPAMNcomposition according to any of claims 50-56, wherein each pegylatedliposome comprises from 1 to 1000 or 30-200 targeting moieties.
 58. TheLp-γPAMN composition according to any of claims 39-57, furthercomprising one or more of an immunostimulatory agent, a detectablemarker and a maleimide, wherein the immunostimulatory agent, thedetectable marker or the maleimide is attached to said PEG or theexterior of the liposome.
 59. The Lp-γPAMN composition according to anyof claims 39-58, wherein the immunostimulating agent is at least oneselected from the group consisting of: a protein immunostimulatingagent; a nucleic acid immunostimulating agent; a chemicalimmunostimulating agent; a hapten; and an adjuvant.
 60. The Lp-γPAMNcomposition of claim 58 or 59, wherein the immunostimulating agent is atleast one selected from the group consisting of: a fluorescein; afluorescein isothiocyanate (FITC); a DNP; a beta glucan; abeta-1,3-glucan; a beta-1,6-glucan; a resolvin (e.g., a Resolvin D suchas D_(n-6DPA) or D_(n-3DPA), a Resolvin E, or a T series resolvin); anda Toll-like receptor (TLR) modulating agent such as, an oxidizedlow-density lipoprotein (e.g. OXPAC, PGPC), and an eritoran lipid (e.g.,E5564).
 61. The Lp-γPAMN composition according to any of claims 58-60,wherein the immunostimulatory agent and the detectable marker is thesame.
 62. The Lp-γPAMN composition according to any of claims 58-61,further comprising a hapten.
 63. The Lp-γPAMN composition of claim 62,wherein the hapten comprises one or more of fluorescein or Beta 1,6-glucan.
 64. The Lp-γPAMN composition according to any of claims 12-63,which further comprises at least one cryoprotectant selected from thegroup consisting of mannitol; trehalose; sorbitol; and sucrose.
 65. Atargeted composition comprising the composition according to any ofclaims 1-64.
 66. A non-targeted composition comprising the compositionaccording to any of claims 1-49.
 67. The Lp-γPAMN composition accordingto any of claims 12-66, which further comprises carboplatin and/orpembroluzumab.
 68. A pharmaceutical composition comprising the liposomalgamma polyglutamated aminopterin composition according to any of claims12-67.
 69. A pharmaceutical composition comprising gamma polyglutamatedaminopterin composition according to any of claims 1-7.
 70. Thecomposition of any of claims 1-69, for use in the treatment of disease.71. Use of the composition of any of claims 1-70, in the manufacture ofa medicament for the treatment of disease.
 72. A method for treating orpreventing disease in a subject needing such treatment or prevention,the method comprising administering the composition of any of claims1-70 to the subject.
 73. A method for treating or preventing disease ina subject needing such treatment or prevention, the method comprisingadministering the liposomal gamma polyglutamated aminopterin compositionof any of claims 12-69 to the subject.
 74. A method of killing ahyperproliferative cell that comprises contacting a hyperproliferativecell with the composition of any of claims 1-69.
 75. A method of killinga hyperproliferative cell that comprises contacting a hyperproliferativecell with the liposomal gamma polyglutamated aminopterin composition ofany of claims 12-69.
 76. The method of claim 74 or 75, wherein thehyperproliferative cell is a cancer cell, a mammalian cell, and/or ahuman cell.
 77. A method for treating cancer that comprisesadministering an effective amount of the composition of any of claims1-69 to a subject having or at risk of having cancer.
 78. A method fortreating cancer that comprises administering an effective amount of theliposomal gamma polyglutamated aminopterin composition of any of claims12-68 to a subject having or at risk of having cancer.
 79. The method ofclaim 77 or 78, wherein the cancer is selected from the group consistingof: a non-hematologic malignancy including such as for example, lungcancer, pancreatic cancer, breast cancer, ovarian cancer, prostatecancer, head and neck cancer, gastric cancer, gastrointestinal cancer,colorectal cancer, esophageal cancer, cervical cancer, liver cancer,kidney cancer, biliary duct cancer, gallbladder cancer, bladder cancer,sarcoma (e.g., osteosarcoma), brain cancer, central nervous systemcancer, and melanoma; and a hematologic malignancy such as for example,a leukemia, a lymphoma and other B cell malignancies, myeloma and otherplasma cell dyscrasias.
 80. The method of claim 77 or 78, wherein thecancer is a member selected from the group consisting of: lung cancer,breast cancer, colon cancer, pancreatic cancer, gastric cancer, bladdercancer, head and neck cancer, ovarian cancer, and cervical cancer. 81.The method of claim 77 or 78, wherein the cancer is a member selectedfrom wherein the cancer is a member selected from head and neck cancer,stomach cancer, osteosarcoma (e.g., osteosarcoma), Non-Hodgkin'slymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides(cutaneous T-cell lymphoma) choriocarcinoma, and chorioadenoma,nonleukemic meningeal cancer, soft tissue sarcoma (desmoid tumors,aggressive fibromatosis, bladder cancer, and central Nervous System(CNS) lymphoma.
 82. The method of claim 77 or 78, wherein the cancer isa member selected from selected from the group consisting of: colorectalcancer, breast cancer, ovarian cancer, lung cancer, head and neckcancer, pancreatic cancer, gastric cancer, and mesothelioma.
 83. Amethod for treating cancer that comprises administering an effectiveamount of the Lp-γPAMN composition of any of claims 50-66 to a subjecthaving or at risk of having a cancer cell that expresses on its surfacea folate receptor bound by the targeting moiety.
 84. A maintenancetherapy for subjects that are undergoing or have undergone cancertherapy that comprise administering an effective amount of thecomposition of any of claims 1-69 to a subject that is undergoing or hasundergone cancer therapy.
 85. A maintenance therapy for subjects thatare undergoing or have undergone cancer therapy that compriseadministering an effective amount of the liposomal gamma polyglutamatedaminopterin composition of any of claims 12-69 to a subject that isundergoing or has undergone cancer therapy.
 86. A method for treating adisorder of the immune system that comprises administering an effectiveamount of the composition of any of claims 1-69 to a subject having orat risk of having a disorder of the immune system, optionally whereinthe disorder of the immune system is selected from: inflammation (e.g.,acute and chronic), systemic inflammation, rheumatoid arthritis,inflammatory bowel disease (IBD), Crohn disease,dermatomyositis/polymyositis, systemic lupus erythematosus, andTakayasu, and psoriasis.
 87. A method for treating a disorder of theimmune system that comprises administering an effective amount of theliposomal gamma polyglutamated aminopterin composition according to anyof claims 8-69 to a subject having or at risk of having a disorder ofthe immune system, optionally wherein the disorder of the immune systemis selected from: inflammation (e.g., acute and chronic), systemicinflammation, rheumatoid arthritis, inflammatory bowel disease (IBD),Crohn disease, dermatomyositis/polymyositis, systemic lupuserythematosus, and Takayasu, and psoriasis.
 88. A method for treating:(a) an infectious disease that comprises administering an effectiveamount of the composition according to any of claims 1-69 to a subjecthaving or at risk of having an infectious disease; (b) an infectiousdisease, cardiovascular disease, metabolic disease, or another disease,that comprises administering an effective amount of the compositionaccording to of any of claims 1-59 to a subject having or at risk ofhaving an infectious disease, cardiovascular disease, or anotherdisease, wherein the disease is a member selected from: atherosclerosis,cardiovascular disease (CVD), coronary artery disease, myocardialinfarction, stroke, metabolic syndrome, a gestational trophoblasticdisease, and ectopic pregnancy; (c) an autoimmune disease, thatcomprises administering an effective amount of the composition accordingto of any of claims 1-59 to a subject having or at risk of having anautoimmune disease; (d) rheumatoid arthritis, that comprisesadministering an effective amount of the composition according to of anyof claims 1-59 to a subject having or at risk of having rheumatoidarthritis; (e) an inflammatory condition that comprises administering aneffective amount of the composition according to of any of claims 1-59to a subject having or at risk of having inflammation, optionallywherein the inflammation is acute, chronic, and/or systemicinflammation; or (f) a skin condition that comprises administering aneffective amount of the composition according to of any of claims 1-59to a subject having or at risk of having a skin condition, optionallywherein the skin condition is psoriasis.
 89. A method for treating aninfectious disease that comprises administering an effective amount ofthe liposomal gamma polyglutamated aminopterin composition according toof any of claims 12-69 to a subject having or at risk of having aninfectious disease.
 90. A method of delivering gamma polyglutamatedaminopterin to a tumor expressing a folate receptor on its surface, themethod comprising: administering the Lp-γPAMN composition of any ofclaims 1-69 to a subject having the tumor in an amount to deliver atherapeutically effective dose of the gamma polyglutamated aminopterinto the tumor.
 91. A method of preparing a gamma polyglutamatedaminopterin composition comprising the liposomal gamma polyglutamatedaminopterin composition of any of claims 12-69, the method comprising:forming a mixture comprising: liposomal components and gammapolyglutamated antifolate in solution; homogenizing the mixture to formliposomes in the solution; and processing the mixture to form liposomescontaining gamma polyglutamated aminopterin.
 92. A method of preparingthe composition of any of claims 12-69 comprising the steps of: forminga mixture comprising: liposomal components and gamma polyglutamatedaminopterin in a solution; homogenizing the mixture to form liposomes inthe solution; processing the mixture to form liposomes entrapping and/orencapsulating gamma polyglutamated aminopterin; and providing thetargeting moiety on a surface of the liposomes, the targeting moietyhaving specific affinity for at least one of folate receptor alpha(FR-α), folate receptor beta (FR-β) and folate receptor delta (FR-δ).93. The method according to claim 92, wherein the processing stepincludes one or more steps of: thin film hydration, extrusion, in-linemixing, ethanol injection technique, freezing-and-thawing technique,reverse-phase evaporation, dynamic high pressure microfluidization,microfluidic mixing, double emulsion, freeze-dried double emulsion, 3Dprinting, membrane contactor method, and stirring.
 94. The methodaccording to claim 92, wherein said processing step includes one or moresteps of modifying the size of the liposomes by one or more of steps ofextrusion, high-pressure microfluidization, and/or sonication